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THE  UNIVERSITY  OF  CHICAGO 
SCIENCE  SERIES 


Editorial  Committee 

ELIAKIM   HASTINGS   MOORE,  Chairman 

JOHN  MERLE  COULTER 

ROBERT  ANDREWS  MILLIKAN 


THE  UNIVERSITY  OF  CHICAGO 
SCIENCE  SERIES,  established  by  the 
Trustees  of  the  University,  owes  its  origin  to 
a  belief  that  there  should  be  a  medium  of  publica- 
tion occupying  a  position  between  the  technical 
journals  with  their  short  articles  and  the  elaborate 
treatises  which  attempt  to  cover  several  or  all 
aspects  of  a  wide  field.  The  volumes  of  the  series 
will  differ  from  the  discussions  generally  appearing 
in  technical  journals  in  that  they  will  present  the 
complete  results  of  an  experiment  or  series  ot 
investigations  which  previously  have  appeared 
only  in  scattered  articles,  if  published  at  all.  On 
the  other  hand,  they  will  differ  from  detailed 
treatises  by  confining  themselves  to  specific  prob- 
lems of  current  interest,  and  in  presenting  the 
subject  in  as  summary  a  manner  and  with  as  little 
technical  detail  as  is  consistent  with  sound  method. 
They  will  be  written  not  only  for  the  specialist 
but  for  the  educated  layman. 


PROBLEMS  OF  FERTILIZATION 


THE  UNIVERSITY  OF  CHICAGO  PRESS 
CHICAGO,  ILLINOIS 


THE  BAKER  &  TAYLOR  COMPANY 

NEW  YORK 


THE  CAMBRIDGE  UNIVERSITY  PRESS 

LOKDON  AKD  EDINBURGH 

THE  MARUZEN-KABUSHIKI-KAISHA 

TOKYO,    OSAKA,    KYOTO,    FUKUOKA,    SENDAI 

THE  MISSION  BOOK  COMPANY 

SHANeRAI 


PROBLEMS    OF 
FERTILIZATION 


By 

FRANK  RATTRAY  LILLIE 

Professor  of  Embryology 
Uni'versity  of  Chicago 


THE  UNIVERSITY  OF  CHICAGO  PRESS 
CHICAGO,  ILLINOIS 


Copyright  igig  By 
The  University  of  Chicago 


All  Rights  Reserved 


Published  May    igig 


Composed  and  Printed  By 

The  University  of  Chicago  Press 

Chicaffo,  Illinois,  U.S.A. 


PREFACE 

The  following  discussion  of  the  problems  of  fertili- 
zation is  an  outgrowth  of  the  writer's  own  studies  in 
this  field.  It  is  an  attempt  to  present  the  actual 
status  of  the  various  problems  in  a  critical  but  not  in  an 
exhaustive  manner,  and  is  thus  to  be  regarded  as  a  point 
of  departure  for  future  work  as  much  as  a  brief  summary 
of  attained  results.  The  necessary  hmits  of  the  volume 
have  imposed  restrictions  which  have  permitted  scant 
justice  to  numerous  excellent  pieces  of  work  with  which 
all  special  students  are  famihar;  for  this  the  absence 
of  textbook  intention  and  style  may  perhaps  be  addi- 
tional excuse.  The  part  of  the  subject  that  deals 
with  the  basis  of  biparental  inheritance  is  purposely 
treated  more  summarily  than  other  problems,  because 
so  much  that  is  really  extraneous  to  the  problem  of  ferti- 
lization proper  is  involved  in  the  discussion. 

The  inevitable  conflict  between  the  strictly  biological 
and  the  physicochemical  methods  of  analysis  of  bio- 
logical problems  emerges  in  typical  form  in  the  problems 
of  fertilization.  It  is  the  writer's  opinion  that  it  will 
long  continue  to  exist,  but  that  there  is  an  ultimate 
reconciliation,  if  not  in  sight,  at  least  in  prospect,  on  logi- 
cal grounds.  An  equal  hospitality  to  the  results  of  both 
methods  of  inquiry  is  therefore  adopted.  The  tendency 
toward  excessive  simplification  of  the  physicochemical 
school  is  constantly  being  checked  by  the  biological 
school,  and  the  conservatism  of  the  latter  has  been  more 
than  once  rudely  shaken  by  the  former.     These  mutual 


Vll 


1972f> 


viii  PROBLEMS  OF  FERTILIZATION 

assaults  suggest  co-operation  such  as  is  carried  out  at  the 
Marine  Biological  Laboratory  of  Woods  Hole.  A  large 
share  of  recent  work  on  the  subject  has  as  a  matter  of 
fact  been  done  in  this  institution,  including  the  writer's 
own  work  and  that  of  numerous  other  authors  cited 
below. 

Chapter  i  is  reprinted  slightly  modified  from  an 
address  delivered  before  the  American  Society  of  Natu- 
ralists and  the  Zoological  Section  of  the  American  Asso- 
ciation for  the  Advancement  of  Science,  December  30, 
1915,  published  in  Science,  N.S.,  Vol.  XLIII,  Jan- 
uary  14,  1916. 


CONTENTS 

PAGE 

List  of  Illustrations ^^ 

CHAPTER 

I.  The  History  of  the  Fertilization  Problem     .     .  i 

II.  The  Place  of  Fertilization  in  the  Life-History   .  31 

HI.  The  Morphology  of  Fertilization 44 

IV.  The  Physiology  of  the  .Spermatozoon  .      .      .      .91 

V.  The  Physiology   of  Fertilization 129 

VI.  The  Problem  of  Specificity  in  Fertilization.     .  1S4 

VII.  The  Problem  of  Activation 227 

Index     .      .     .  ■ -75 


IX 


LIST  OF  ILLUSTRATIONS 


PAGE 


Fig.    I.     Spermatozoa  of  Various  Animals      ....       49 

Fig.    2.    Drawings  from  Photographs  of  Nereis  Eggs 

IN  A  Suspension  of  India  Ink  in  Sea-Water      52 

Fig.    3.    Penetration  of  the  Spermatozoon  in  the  Egg 

OF  Nereis,  from  Sections 54 

Fig.  4.  Spermatozoon  in  the  Oligochaete  Rhynchelmis, 
in  the  Egg  of  the  Bat  Vespertilio  nockda, 

AND      IN      THE      EgG      OF      THE      SnAIL     Pliysa 

fontinalis 56 

Fig.    5.    The  Formation  of  the  Fertilization  Mem- 
brane IN  THE   Egg   of  the   Sea   Urchin 
Strongylozenlrotus  pur  pur  at  us 58 

Fig.  6.  Entrance  of  Spermatozoon  and  Formation 
OF  the  Fertilization  Membrane  in  Ascaris 
megalocephala        59 

Fig.    7.    Fertilization  of  the  Egg  of  the  Sea  Urchin 

Toxopneustes 61 

Fig.  8.  Sections  of  Successive  Stages  Showing  the 
Internal  Phenomena  of  Fertilization  in 
Nereis         62-63 

Fig.    9.     Development    of    Ascaris    megalocephala,    and 

Plerotrachea  at  Various  Stages      ....       66 

Fig.  to.     Fertilization    of    a    Nematode    (Ancyracan- 

ihus  cystidicola) 69 

Fig.  II.  Effects  of  Centrifugal  Force  on  Penetra- 
tion of  the  Spermatozoon  in  A^crm    ...       72 

Fig.  12.    Horizontal  Section  of  the   Germinal  Disk 

OF  the  Pigeon's  Egg 79 

Fig.  13.     Photograph  of  the  Aggregation  of  a  Sperm 

Suspension  of  Nereis 95 


XI 


xii  PROBLEMS  OF  FERTILIZATION 


PAGE 


Fig.  14.  Reaction  of  a  Sperm  Suspension  of  Nereis 
TO  A  Drop  of  i  Per  Cent  C02  Sea- 
Water       105 

Fig.  15.  Diagram  of  the  Reaction  of  Spermatozoa  of 
Nereis  to  a  Drop  of  i  Per  Cent  C02  in  Sea- 
Water       106 

Fig.  16.     Curve  of  Fertilizing  Power  of  Perfectly 

Fresh  Sperm  Suspensions  of  Arhacia      .     .     135 

Fig.  17.  Curve  of  Fertilizing  Power  of  Sperm  Sus- 
pensions OF  Arhacia  About  20  Minutes  Old     137 

Fig.  18.    Imbibition    of    Water    by    Arhacia    Eggs    in 

Diluted  Sea-Water 150 

Fig.  19.     Curve  of   Fertilization  Superimposed  upon 

Butyric  Acid  Treatment  in  ylrftac/a       .      .     168 


CHAPTER  I 
THE    HISTORY    OF    THE    FERTILIZATION    PROBLEM 

The  two  primary  interests  and  compelling  motives 
of  mankind  have  always  been  hunger  and  love— the  in- 
stincts of  self-preservation  and  race-preservation.     The 
reasoning   faculty   of   man  early    turned    with    eager 
interest    to    the    great   problem    of    reproduction,    but 
for  a  long  time  without  attaining  results  that  could 
be  called  scientific;    and  it  was  not  until  well  into  the 
nineteenth  century  that  the  problems  connected  with 
sexual  reproduction  could  be  scientifically  formulated 
by   separating   the   problem    of    fertilization   from    all 
its  extraneous  surroundings.     In  early  human  culture 
reproduction    received    its    only    interpretation   at   the 
hands  of  priests  and  mystery  men;   its  first  philosoph- 
ical and  scientific  treatment  was  one  of  the  distinctions 
of  the  Greeks,  especially  of  that  great  philosopher  and 
father  of  science,  Aristotle,  who  combined  observation 
and  reflection  in  the  interpretation  of  nature.     Aristotle 
devoted  a  separate  treatise,  which  has  come  down  to  us, 
to  animal  reproduction.     Among  other  things  he  studied 
the  development  of  the  chick  day  by  day  with  so  much 
detail  that  Harvey  felt  impelled  to  say,   1,900  years 
later:    "Aristotle  among  the  ancients,  and  Hieronymus 
Fabricius  of  Aquapendente  among  the  moderns,  have 
written  with  so  much  accuracy  on  the  generation  and 
formation  of  the  chick  from  the  egg  that  little  seems 
left  for  us  to  do. "  mmtkM-- 


^C«|» 


2  PROBLEMS  OF  FERTILIZATION 

From  the  time  of  the  Greeks  to  that  of  Harvey 
(165 1)  there  was  but  little  progress  in  the  knowledge  of 
reproduction  and  none  in  the  theory,  as  will  appear 
from  the  views  of  Aristotle,  the  current  views  of  medical 
men  of  Harvey's  time,  and  of  Harvey  himself.  Aristotle 
says : 

The  male  is  the  efficient  agent,  and  by  the  motion  of  his  genera- 
tive virtue  (genitura),  creates  what  is  intended  from  the  matter 
contained  in  the  female;  for  the  female  always  supplies  the  matter, 
the  male  the  power  of  creation,  and  this  it  is  which  constitutes 
one  male,  another  female.  The  body  and  the  bulk,  therefore, 
are  necessarily  supplied  by  the  female;  nothing  of  the  kind  is  re- 
quired from  the  male;  for  it  is  not  even  requisite  that  the  instru- 
ment, nor  the  efficient  agent  itself,  be  present  in  the  thing  that  is 
produced.  The  body  then  proceeds  from  the  female,  the  vital 
principle  (anima)  from  the  male;  for  the  essence  of  every  body  is 
its  vital  principle  (anima). ^ 

With  more  common  sense,  if  with  less  metaphysical 
subtlety,  the  physicians  of  the  Middle  Ages  held,  accord- 
ing to  Harvey,  that  conception  is  due  to  a  mingling  of 
male  and  female  seminal  fluids,  ''the  mixture  having 
from  both  equally  the  faculty  of  action  and  the  force  of 
matter;  and  according  to  the  predominance  of  this  or 
that  geniture  does  the  progeny  turn  out  male  or  female" 
(quoted  from  Harvey,  Ex.  32). 

Harvey's  observations  contained  much  that  was  new 
and  significant,  and  the  facts  that  he  discovered  were 
inconsistent  both  with  Aristotle's  ideas  and  with  those 
of  the  physicians.  They  were,  however,  inadequate  for 
sound  generalization. 

Wandering  between  two  worlds,  one  dead 
The  other  powerless  to  be  born, 

^  De  Gen.  Anim.  ii.  4,  quoted  from  Harvey,  "On  the  Generation  of 
Animals,"  Ex.  29. 


HISTORY  OF  THE  FERTILIZATION  PROBLEM       3 

he  descended  deeper  into  the  slough  of  metaphysics  than 
Aristotle,  and  committed  himself  to  the  fantastic  idea 
that  conception  in  the  uterus  is  identical  with,  or  at 
least  analogous  to,  conception  in  the  brain;  and  that  the 
ovum  is  the  product  of  such  unconscious  uterine  desire 
or  conception,  and  receives  no  material  substratum  from 
the  male!'  The  theory  of  reproduction  was  no  whit 
more  advanced  in  the  middle  of  the  seventeenth  century 
than  in  the  time  of  Aristotle. 

The   use   of   the  microscope   in  biological   research 
began  in  the  seventeenth  century;   it  was  the  improve- 

^  "Since  there  are  no  manifest  signs  of  conception  before  the  uterus 
begins  to  relax,  and  the  white  fluid  or  slender  threads  (like  the  spider's 
web)  constituting  the  'primordium'  of  the  future  'conception'  or  ovum, 
shows  itself;  and  since  the  substance  of  the  uterus,  when  ready  to  con- 
ceive, is  very  like  the  structure  of  the  brain,  why  should  we  not  suppose 
that  the  function  of  both  is  similar,  and  that  there  is  excited  by  coitus 
within  the  uterus  a  something  identical  with,  or  at  least  analogous  to, 
an  'imagination'  (phantasma)  or  a  'desire'  (appetitus)  in  the  brain, 
whence  comes  the  generation  or  procreation  of  the  ovum.  For  the  func- 
tions of  both  are  termed  'conceptions,'  and  both,  although  the  primary 
sources  of  every  action  throughout  the  body,  are  immaterial,  the  one  of 
natural  or  organic,  the  other  of  animal  actions;  the  one  (viz.,  the  uterus) 
the  first  cause  and  beginning  of  every  action  which  conduces  to  the 
generation  of  the  animal,  the  other  (viz.,  the  brain)  of  every  action  done 
for  its  preservation.  And  just  as  a  'desire'  arises  from  a  conception  of 
the  brain,  and  this  conception  springs  from  some  external  object  of  de- 
sire, so  also  from  the  male,  as  being  the  more  perfect  animal,  and,  as  it 
were,  the  most  natural  object  of  desire,  does  the  natural  (organic) 
conception  arise  in  the  uterus,  even  as  the  animal  conception  does  in  the 
brain. 

"From  this  desire  or  conception,  it  results  that  the  female  produces 
an  offspring  like  the  father.  For  just  as  we,  from  the  conception  of  the 
'form'  or  'idea'  in  the  brain,  fashion  in  our  works  a  form  resembling  it, 
so,  in  like  manner,  the  'idea'  or  'form'  of  the  father  existing  in  the  uterus 
generates  an  offspring  like  himself  with  the  aid  of  the  formative  faculty, 
impressing,  however,  on  its  work  its  own  immaterial  'form'"  (from 
William  Harvey,  "On  Conception,"  1651). 


4  PROBLEJNIS  OF  FERTILIZATION 

ment  of  this  new  instrument  of  investigation  and  its 
application  to  the  study  of  the  reproductive  substances 
that  furnished  the  first  fundamental  advance  in  the 
theory  of  reproduction  at  the  hands  of  Leeuwenhoek, 
viz.,  the  discovery  of  the  spermatozoa^  in  1677. 

This  discovery  aroused  the  greatest  interest  in  scien- 
tific circles;  a  number  of  investigators  repeated  the 
observations  and  a  spirit  of  speculation  which  led  to 
wild  flights  of  the  imagination  was  aroused.  Leeuwen- 
hoek  had  soon  to  defend  his  priority  in  the  matter  and 
to  protest  against  certain  very  imaginative  views. 
Thus  in  a  letter  dated  June  9,  1699,^  he  defends  his  pri- 
ority and  combats  the  notion  that  the  human  form  can 
be  observed  in  the  spermatozoa.     He  inveighs  especially 

'  This  discovery  is  sometimes  credited  to  Hamm,  described  as  a 
student  of  Leeuwenhoek's.  The  latter  himself  describes  the  occurrence 
as  follows  {Phil.  Trans.,  1678,  containing  a  letter  from  Leeuwenhoek 
dated  November,  1677) :  A  certain  Professor  Cranen,  who  had  frequently 
visited  Leeuwenhoek  for  microscopical  demonstrations,  requested  by  let- 
ter that  he  should  give  Dominus  Hamm,  a  relative  of  his,  some  demon- 
strations of  his  observations.  On  his  second  visit  D.  Hamm  brought 
in  a  glass  vial  some  seminal  fluid  and  stated  that  he  had  observed 
living  animals  in  it;  Leeuwenhoek  confirmed  this  observation  and 
repeated  it  many  times.  In  this  letter  he  gives  a  fair  description  of  the 
spermatozoa,  their  form,  size,  and  movements,  and  stated  that  he  had 
observed  them  three  or  four  years  previously  and  mistaken  them  for 
globules.  He  did  not  at  this  time  speculate  as  to  the  meaning  of  the 
spermatozoa,  but  in  true  scientific  spirit  began  to  make  comparative 
obser\-ations,  and  in  1678  he  described  and  figured  spermatozoa  of  the 
rabbit  and  frog  among  others. 

The  credit  of  this  discovery  seems  to  me  to  belong  rightly  to  the 
investigator  whose  wide  experience  in  the  field  of  microscopical  anatomy 
and  whose  scientific  acumen  enabled  him  to  grasp  the  possible  signifi- 
cance of  the  discovery,  not  to  the  chance  observer  who  called  Leeuwen- 
hoek's attention  anew  to  the  subject. 

'  Phil.  Trans.,  Vol.  XXI. 


HISTORY  OF  THE  FERTILIZATION  PROBLEM       5 

against  a  certain  Dr.  Dalen  Patius,  who  claimed  to  have 
seen  the  human  form,  'Hhe  two  naked  thighs,  the  legs, 
the  breast,  both  arms,  etc.,  the  skin  being  pulled  up 
somewhat  higher  did  cover  the  head  like  a  cap. " 

Leeuwenhoek  states  that  he  can  find  nothing  of  the 
sort,  but  he  adds: 

I  put  this  down  as  a  certain  truth,  that  the  shape  of  the 
human  body  is  included  in  an  animal  of  the  masculine  seed; 
but  that  a  man's  reason  shall  dive  or  penetrate  into  this  mystery 
so  far,  that  in  anatomizing  one  of  these  animals  of  the  masculine 
seed  we  should  be  able  to  discover  the  entire  shape  of  the  human 
body,  I  can  not  comprehend. 

In  a  letter  dated  tw^o  weeks  later  he  distinguishes 
two  sorts  of  these  animalcules,  and  concludes  that  the 
one  sort  is  male  and  the  other  female. 

In  France,  in  the  year   1694,  Nicholas  Hartsoeker 
claimed  to  have  been  the  first  to  have  discovered  the 
spermatozoa,    more     than     twenty    years    previously, 
although  he  did  not  publish  until   1678,  a  year  later 
than    Leeuwenhoek's    pubHcation.     Hartsoeker's    ideas 
are  characterized  by  a  high  degree  of  precision.     He 
believes  that  each  spermatozoon  conceals  beneath  its 
''tender  and  delicate  skin"  a  complete  male  or  female 
animal.     The  egg  is  merely  a  source  of  nourishment 
for    the    real    germ    contained    in    the    spermatozoon. 
In  birds  the  spermatozoon  enters  an  egg  to  be  nourished; 
there  is  but  a  single  opening  in  the  egg,  situated  over  the 
so-called  germ,  and  this  opening  closes  after  a  single 
spermatozoon  is  admitted;  but  if  two  spermatozoa  enter 
they  unite  and  form  a  double  monster.     In  mammals 
the  tail  of  the  spermatozoon  is  the  umbilical  cord;  this 
unites  with  the  ovum,  i.e.,  the  placenta,  and  the  latter 


6  PROBLEMS  OF  FERTILIZATION 

with  the  uterus.  Each  one  of  the  male  animals  (sperma- 
tozoa) incloses  an  infinity  of  other  animals  both  male  and 
female,  which  are  correspondingly  small,  and  these  male 
animals  inclose  yet  other  males  and  females  of  the 
same  species,  and  so  forth  in  a  series  which  includes 
all  the  members  of  the  species  which  are  to  be  produced 
up  to  the  end  of  time.  No  difficulty  was  found  in  this 
conception,  for  the  atomic  theory  of  matter  was  not 
yet  placed  on  a  scientihc  basis. 

Thus  was  founded  and  flourished  for  its  brief  day  the 
school  of  the  spermatists.  Unhampered  by  any  scien- 
tific conception  of  matter,  hving  or  non-living,  there  was 
no  obstacle  to  the  eye  of  faith  and  no  impediment  to 
the  age-old  longing  to  make  an  intelligible  universe  out 
of  the  scraps  of  experience. 

In  the  entire  eighteenth  century,  although  specula- 
tion continued  rife,  there  was  only  one  notable  contribu- 
tion to  our  subject.  This  was  the  work  of  the  Abbe 
Spallanzani,  Experiences  pour  servir  a  Vhistoire  de  la 
generation  des  animaux  et  des  plantes,  published  in  Geneva 
in  1785.  His  woj-king  hypotheses  were  naturally  in 
the  spirit  of  the  times.  Theories  of  reproduction, 
he  says,  may  be  reduced  to  two. 

The  one  explains  the  development  of  organisms  mechanically, 
the  other  supposes  them  to  pre-exist,  and  waiting  only  for  fer- 
tilization to  develop  them.  The  second  system  has  given  birth 
to  two  different  parties,  one  beheving  that  the  organism  is  pre- 
formed in  the  ovum,  the  other  that  it  is  performed  in  the 
spermatozoon. 

Spallanzani  believed  that  his  observations  destroyed 
the  epigenetic  theory  as  propounded  by  BuiTon  and  others, 
because  they  demonstrated  the  existence  of  the  "fetuses " 


HISTORY  OF  THE  FERTILIZATION  TROBLEM       7 

(ova)  in  the  females  of  toads,  frogs,  and  salamanders, 
prior  to  the  act  of  fertilization,  which  according  to  the 
epigenesists  animates  or  creates  the  germ.  For  the 
same  reason  the  spermatists  must  also  be  wrong.  Spal- 
lanzani  thus  combated  epigenesis  as  understood  in  the 
eighteenth  century,  and  also  the  ideas  of  the  spenna- 
tists,  and  he  was  led  to  deny  that  spermatozoa  are  neces- 
sary for  fertilization,  and  to  hold  that  the  fertilizing 
power  of  the  seminal  fluid  resides,  not  in  the  spermatozoa, 
but  in  the  fluid  medium  that  accompanies  them;  and 
this  in  spite  of  the  fact  that  his  final  experiments  really 
proved  the  reverse. 

His  work  contains  a  great  wealth  of  observation  and 
experiment,  so  that  it  will  be  possible  merely  to  indicate 
some  of  his  chief  results.  In  the  first  place  he  demon- 
strated that  in  frogs  and  toads  fertilization  takes  place 
outside  of  the  body,  and  for  the  first  time  he  success- 
fully carried  out  artificial  insemination,  thus  laying  the 
foundation  for  the  artificial  propagation  of  many  ani- 
mals. In  making  these  experiments  he  thought  he  found 
cases  in  which  seminal  fluid  devoid  of  spermatozoa  would 
fertilize  and  thus  fell  into  the  error,  which  he  was  so 
ready  to  accept  from  his  opposition  to  the  spermatists, 
that  the  fluid  medium  of  the  seminal  fluid  was  the  ferti- 
lizing substance.  He  also  investigated  the  conditions 
of  successful  insemination,  with  reference  to  the  duration 
of  fertilizing  power,  exposure  to  various  chemicals,  to 
heat,  etc.  The  amount  of  dilution  of  which  the  semi- 
nal fluid  was  capable  was  likewise  carefully  investigated. 
By  experiment  he  excluded  the  idea  that  fertiHzation 
might  be  an  efi'ect  of  an  emanation,  or  vapor,  arising 
from  the  sperm. 


8  PROBLEMS  OF  FERTILIZATION 

He  concluded  that  the  seminal  fluid  acts  by  accel- 
erating the  vital  processes;  it  enters  the  body  through 
pores  and  stimulates  the  action  of  the  heart.  This  idea 
offered  no  difficulty  to  one  who  believed  that  the  organ- 
ism was  preformed  in  the  ovum,  and  it  was  supported 
by  the  observation  that  the  beating  of  the  heart  was  the 
first  observable  movement  of  the  embryo.  Bonnet 
suggested  to  him  the  problem:  If  the  spermatic  fluid 
might  stimulate  the  heart  of  the  embryo  in  the  process 
of  fertilization,  why  might  not  other  fluids  produce  the 
same  effect?  He  was  thus  led  to  attempt  the  first 
experiments  on  artificial  parthenogenesis;  he  tried  to 
start  the  development  of  eggs  by  electricity,  by  the 
action  of  extracts  of  all  the  various  organs,  by  vinegar, 
dilute  alcohol,  lemon  juice,  and  other  substances,  all 
without  effect. 

It  is  interesting  to  see  how  his  experiments  led  to 
hypotheses,  and  these,  even  though  wrong,  to  further 
experiments,  some  of  which,  like  his  experiments  on 
artificial  parthenogenesis,  were  not  taken  up  again  in 
a  fruitful  way  for  over  a  century. 

His  final  experiments  are  those  so  often  quoted  as 
furnishing  the  proof  that  fertilizing  power  resides  in  the 
spermatozoon.  He  showed  that,  if  diluted  sperm  be 
filtered  through  a  sufficient  number  of  layers  of  filter 
paper,  the  filtrate  has  no  fertilizing  power,  whereas  the 
residue  washed  off  the  filter  paper  will  fertilize.  But 
he  did  not  himself  draw  the  correct  conclusion;  he 
says  the  experiment  proves  '^that  filtration  removes 
from  spermatized  water  its  fertilizing  power,  inasmuch 
as  the  seminal  fluid  which  was  contained  in  it  remains 
on  the  filter  papers,  from  which  one  can  extract  it  by 


HISTORY  OF  THE  FERTILIZATION  PROBLEM        9 

pressing  them."  It  is  perfectly  clear  that  Spallanzani 
himself  never  held  that  the  spermatozoa  themselves 
were  the  fertilizing  agents,  but,  on  the  contrary,  he 
contests  this  idea  strongly  as  leading  to  spermatist 
delusions. 

After  Spallanzani  there  was  no  real  advance  in  the 
theory  of  fertilization  until  the  publication  of  Prevost 
and  Dumas'  New  Theory  of  Reproduction  in  1824.  They 
observed  that  young  animals  incapable  of  breeding, 
old  animals  beyond  the  breeding  stage,  the  infertile 
mule,  and  birds  outside  of  the  breeding  season  possess 
no  spermatozoa,  and  they  conclude  that  these  facts 
"sufficiently  prove  the  importance  of  the  animalcules, 
and  show  that  there  exists  an  intimate  relationship  be- 
tween their  presence  in  the  reproductive  organs  and 
the  fertilizing  power  of  the  animal."  In  a  long  series 
of  experiments  they  investigated  the  conditions  of 
fertilization  in  frogs:  all  conditions  that  destroy  the 
animalcules  destroy  also  the  fertilizing  power  of  sperm 
suspensions ;  the  filtrate  of  a  sperm  suspension  devoid  of 
spermatozoa  will  not  fertilize;  the  redissolved  residue 
of  a  suspension  evaporated  to  dryness  will  not  fertilize, 
etc. ;  the  number  of  eggs  fertilized  is  always  less  than  the 
number  of  "animalcules"  employed.  They  came  to 
the  conclusion,  therefore,  that  "the  prolific  principle 
resides  in  the  spermatic  animalcules." 

In  their  subsequent  publications  they  concluded 
that  it  is  "infinitely  probable  that  the  number  of  animal- 
cules employed  in  fertilization  corresponds  to  that  of 
the  embryos  developed  ....  so  that  the  action  of 
these  animalcules  which  we  regard  as  the  male  repro- 
ductive elements  is  individual,  not  collective." 


lo  PROBLEMS  OF  FERTILIZATION 

They  concluded  that  a  spermatozoon  penetrates 
each  egg  and  becomes  ^'the  rudiment  of  the  nervous 
system,  and  that  the  membrane  (germ  disk  of  the  egg) 
in  which  it  is  implanted  furnishes,  by  the  diverse 
modifications  which  it  undergoes,  all  the  other  organs  of 
the  embryo. " 

These  studies  gave  a  new  impetus  to  the  study  of 
fertilization;  some  were  convinced  that  Prevost  and 
Dumas  were  essentially  correct,  w^hile  others  still  adhered 
to  the  idea  that  the  fluid  part  of  the  seminal  fluid  was 
the  fertilizing  medium.  Thus  the  celebrated  embry- 
ologist  Bischoff  in  1842  does  not  hesitate  to  declare 
outright  for  the  latter  view,  ''that  only  the  dissolved 
part  of  the  semen  penetrates  into  the  egg  and  thus 
completes  fertilization. "     He  considered  that 

Valentin's  hypothesis  united  all  the  facts:  the  seminal  fluid  is  so 
unstable  chemically  as  to  break  down  as  soon  as  the  particles 
come  to  rest;  it  is  similar  to  the  blood  in  this  respect,  but  it  is  not 
in  regular  circulation  and  the  function  of  maintaining  its  chemical 
composition  is  relegated  to  the  movements  of  the  spermatozoa. 

However,  Bischoff  subsequently  became  convinced 
that  the  spermatozoa  were  themselves  the  essential 
agents,  though  he  still  refused  to  believe  in  the  pene- 
tration of  the  egg.  Kolliker  had  put  forward  a  contact 
theory  of  fertilization,  which  Bischoff  regarded  merely 
as  a  statement  of  facts  requiring  further  development. 
He  therefore  adopted  the  idea  of  catalyzers,  at  that 
time  a  new  idea  in  chemistry,  and  held  that  the  sperma- 
tozoon was  essentially  a  catalytic  agent,  i.e.,  as  he  defined 
it,  "a,  form  of  matter  characterized  by  definite  trans- 
formation and  internal  movement"  which  it  transmits 
by  contact  to  the  egg,  which  is  in  a  condition  of  maximum 


HISTORY  OF  THE  FERTILIZATION  PROBLEM     1 1 

tension  or  inclination  to  assume  the  same  form  of  trans- 
formation and  movement.  Fertilization  is  thus  not 
a  process  of  union  and  fusion  as  in  ordinary  chemical 
combination,  but  a  catalytic  process,  as  defined  above. 

This  point  of  view  deserves  to  be  emphasized  as  one 
of  the  first  attempts  at  a  physicochemical  explanation 
of  fertilization. 

For  some  time  naturalists  were  divided  between  the 
two  points  of  view,  viz.,  that  of  Prevost  and  Dumas, 
that  the  sperm  penetrated  into  the  egg,  and  that  of 
Kolliker  and  Bischoff,  that  it  acted  by  contact.  Lalle- 
mand  (1841)  well  expresses  the  view  of  those  who  be- 
Heved  in  the  union  of  the  ovum  and  spermatozoon. 

Each  of  the  sexes  furnishes  material  already  organized  and 

living A  fluid  obviously  can  not  transmit  form  and  life 

which  it  does  not  possess Fertilization  is  the  union  of  two 

living  parts  which  mutually  complete  each  other  and  develop  in 

common When  one  embraces  in  a  single  point  of  view  the 

reproduction  of  all  living  beings,  one  arrives  at  the  following  more 
general  formula:  Reproduction  is  the  separation  of  a  living  part 
which  may  either  develop  separately  or  acquire  from  another  living 
part  the  supplementary  elements  necessary  for  the  ulterior  develop- 
ment of  a  being  similar  to  the  type The  preservation  of 

the  type  is  due  to  the  extension  of  the  same  act  which  has  produced 
the  development  of  each  individual  being. 

This  is  the  most  complete  statement  of  the  principle 
of  genetic  continuity  that  I  have  found  in  the  Hterature 
of  this  period. 

These  observations  and  conclusions  were  found  on 
the  eve  and  early  morrow  of  the  great  biological  general- 
ization, the  cell  theory.  Though  Schwann  interpreted 
the  ovum  as  a  cell  (1838),  this  view  did  not  at  once 
become  dominant  and  was  generally  accepted  only  after 


12  PROBLEMS  OF  FERTILIZATION 

over  twenty  years  of  discussion.  The  view  that  sperma- 
tozoa were  parasitic  organisms  was  more  or  less  current 
until  Kolliker  in  1841  showed  by  their  development 
that  they  were  modified  cells.  Nevertheless,  there  was, 
strictly  speaking,  no  immediate  application  of  these 
results  to  the  problems  of  fertilization. 

The  half-century  from  1824  to  1874  yielded  relatively 
little  advance  in  fertilization  theory;  the  opinion  that 
the  spermatozoon  actually  penetrated  into  the  ovum 
gradually  gained  ground,  largely  from  the  very  logic  of 
the  situation,  but  partly  from  various  observations. 
Bischoff's  contact  theory,  which  was  the  only  alternative, 
was  criticized,  because  if  the  sperm  does  not  penetrate, 
but  remains  outside  of  the  membrane,  there  is  absence  of 
that  direct  contact  between  sperm  and  egg  substance 
postulated  by  the  theory.  Wagner's  criticism  was  also 
very  effective;  a  ferment  does  not  determine  the  char- 
acter of  a  reaction,  but  the  spermatozoon  does,  for  it 
transmits  paternal  characteristics.  In  the  way  of  ob- 
servations Barry  (1840),  Newport'  (1854-55),  Meissner, 
(1855),  and  others  maintained  observations  of  penetra- 
tion of  the  ovum  by  the  spermatozoon;  Keber  (1854) 
laid  especial  emphasis  on  the  micropyle  as  adapted  for 
entrance  of  a  spermatozoon.  These  observations  were 
on  the  whole  inconclusive,  for  actual  penetration  was 
not  observed  but  inferred  from  the  presence  of  spermato- 
zoa inside  the  egg  membrane.  Moreover,  the  spermato- 
zoon could  not  be  discovered  within  the  egg. 

'  Newport's  observations  rose  to  a  higher  plane  than  those  of  the 
others,  for  he  actually  observed  in  the  frog's  egg  (i)  that  the  first 
plane  of  cleavage  is  in  line  with  the  point  on  the  egg  artificially  impreg- 
nated, (2)  that  it  marks  the  plane  of  symmetry  of  the  embryo,  (3) 
that  the  head  of  the  young  frog  is  turned  toward  the  same  point. 


HISTORY  OF  THE  FERTILIZATION  PROBLEiM     13 

The  modern  period. — The  preceding  period  (1824-74) 
was  coincident,  as  we  have  seen,  with  the  early  history 
of  the  cell  theory,  but  the  demonstration  of  the  uni- 
cellular character  of  the  ovum  and  spermatozoon 
had  little  effect  upon  the  problems  of  fertilization.  'Jlie 
cell  theory  was  still  incomplete ;  the  free  formation  of  the 
nuclei  was  still  held  by  competent  naturaHsts,  and 
nothing  was  known  of  the  phenonema  of  karyokinesis. 
The  cytological  investigations  of  the  next  ten  years 
(1874-84)  were  destined  to  lay  the  foundations  of  the 
modern  nuclear  theory  in  its  broad  outlines.  The 
fertilization  studies  of  this  period  were  mainly  morpho- 
logical, and  while  it  is  correct  to  say  that  they  were  largely 
dominated  by  the  growing  nuclear  theory  it  is  also  strictly 
true  that  they  contributed  in  no  small  measure  to  its 
upbuilding.  Though  the  penetration  of  the  sperma- 
tozoon into  the  egg  had  long  been  suspected,  it  was  first 
clearly  demonstrated  at  this  time;  the  origin  of  the  egg 
nucleus  by  two  successive  divisions  of  the  germinal  vesicle 
was  discovered;  the  origin  of  the  sperm  nucleus  from  the 
head  of  the  spermatozoon,  the  sperm  aster,  the  union  of 
the  egg  nucleus  and  the  sperm  nucleus,  the  relation  of 
these  to  the  first  cleavage  spindle,  the  origin  of  the  ferti- 
lization membrane,  the  ill  effects  of  polyspermy  and  the 
theory  of  its  prevention,  and  finally  the  doctrine  of  the 
equivalence  of  the  egg  and  sperm  nuclei,  and  the  bipar- 
ental  character  of  the  nuclei  of  sexually  produced  organ- 
isms, as  first  laid  down  by  Van  Beneden,  were  products 
of  the  period  also.  No  period  of  cytological  research 
seems  to  me  of  greater  significance  than  this. 

There  was  almost  a  complete  cessation  of  investi- 
gation from  1855  to  1873,  when  the  dawn  of  the  modern 


14  PROBLEMS  OF  FERTILIZATION 

period  broke  suddenly.  In  1873  Biitschli  observed  in 
the  egg  of  a  nematode  the  approach  and  contact  of  the 
two  structures,  which  we  now  know  to  be  the  germ  nuclei, 
immediately  preceding  the  first  cleavage  of  the  ovum. 
But  no  interpretation  was  presented.  In  1874  Auer- 
bach  described  the  appearance  of  two  nuclei  at  opposite 
ends  of  the  elongated  egg  of  Rhabdites;  these  increase 
in  size,  migrate  toward  the  center  of  the  egg,  meet, 
rotate  through  90°,  and  fuse  together.  A  dicentric 
figure  appears  and  cleavage  follows.  What  is  the  origin 
of  these  two  nuclei  and  the  significance  of  their  union  ? 
The  fusion  of  two  nuclei  was  at  the  time  entirely  without 
analogy.  Auerbach  states:  ''It  is  natural  to  assume 
that,  as  for  the  reproduction  of  organisms  the  copulation 
of  two  individuals,  or  at  least  of  two  cells  in  some  form 
or  other,  is  so  frequently  necessary,  so  here  a  similar 
condition  is  found  for  nuclear  reproduction." 

Auerbach  supposes  the  two  nuclei  which  appear  at 
opposite  ends  of  the  elongated  egg  to  have  arisen  freely; 
one  of  these  comes  from  the  end  where  the  sperma- 
tozoa had  penetrated,  the  other  from  the  opposite 
end,  where  the  germinal  vesicle  had  disappeared.  The 
difference  of  origin  influences  the  quality  of  the  nuclear 
materials  arising,  according  to  his  conception,  de  novo; 
fusion  of  the  nuclei  counteracts  the  differences  thus  aris- 
ing; but  all  this  would  be  undone  if  the  division  of  the 
fusion  nucleus  followed  along  the  plane  of  the  union; 
hence  the  rotation  through  90°. 

In  the  next  year  Biitschli  again  observed  fusion  of 
nuclei  in  nematode  eggs  before  the  first  cleavage. 
However,  he  did  not  accept  Auerbach's  interpretation, 
but  he  tended  to  regard  it  as  a  general  law  of  nuclear 


HISTORY  OF  THE  FERTILIZATION  TROBLEM     15 

formation  that  lirst  two  or  several  small  nuclei  arise, 
and  subsequently  fuse;  this  he  finds  to  occur  even  in 
the  blastomeres  of  the  four-  and  eight-cell  stages. 

About  the  same  time  (1875)  Van  Beneden  also 
observed  similar  phenomena  in  the  rabbit's  egg.  lie 
did  not  see  spermatozoa  enter  the  egg,  but  he  found  them 
with  their  heads  closely  applied  to  the  surface  in  every 
unsegmented  egg,  and  came  to  the  conclusion  that 
fertilization  consisted  essentially  in  fusion  of  the  sper- 
matic substance  with  the  superficial  layer  of  the  vitellus. 
At  a  little  later  stage  he  found  a  small  nucleus  in  the 
cortical  layer  of  the  egg;  this  he  called  the  peripheral 
pronucleus;  a  central  pronucleus  appeared  simultane- 
ously. They  grow,  approach  one  another,  and  meet 
in  the  center.  Later  there  is  only  one  nucleus,  probably 
formed  by  the  union  of  the  two. 

As  I  have  shown  that  the  spermatozoa  attach  to  the  surface 
of  the  vitellus  and  mix  with  its  superficial  layer,  it  appears  probable 
to  me  that  the  superficial  pronucleus  is  formed,  partially  at  least, 
at  the  expense  of  the  spermatic  substance.  If,  as  I  think,  the 
central  pronucleus  is  constituted  of  elements  furnished  by  the  egg, 
the  first  nucleus  of  the  embryo  would  be  the  result  of  union  of  male 
and  female  elements.  I  put  forth  this  latter  idea  simply  as  a 
hypothesis,  an  interpretation  which  may  or  may  not  be  accepted. 

The  way  was  now  clear  for  the  definitive  solution  of 
the  old  riddle  of  the  relation  of  the  egg  and  spermatozoon 
which  was  quickly  furnished  by  O.  Her  twig  and  Hermann 
Fol.  The  observations  of  these  authors  appear  to  have 
been  made  independently  and  nearly  sunultaneously. 
In  1875  Hertwig  observed  and  described  correctly  the 
principal  phenomena  of  fertihzation  in  the  sea  urchin 
egg.     He  did  not  actually  see  the  penetration  of  the 


1 6  PROBLEMS  OF  FERTILIZATION 

spermatozoon,  but  he  observed  the  sperm  nucleus  and 
its  aster  so  soon  after  that  he  had  no  doubt  of  the 
correct  interpretation;  he  also  observed  the  approach 
of  the  sperm  nucleus  and  the  egg  nucleus  to  the  center 
of  the  egg  and  their  apparent  fusion. 

Fertilization  has  been  previously  interpreted  as  a  fusion  of 
two  cells,  but  we  have  now  seen  that  the  most  important  process 
involved  is  the  fusion  of  the  two  nuclei.  The  union  of  the  egg 
nucleus  with  the  sperm  nucleus  is  necessary  to  produce  a  nucleus 
endowed  with  living  forces  adequate  effectively  to  stimulate  the 
later  developmental  processes  in  the  yolk,  and  to  control  them  in 
many  respects. 

Fol's  observations,  made  partly  independently  of 
Hertwig's  and  partly  after  the  publication  of  Hertwig's 
first  paper,  supplemented  Hertwig's  in  several  important 
respects:  (i)  He  observed  the  details  of  penetration  of 
the  spermatozoon  with  a  clearness  that  has  never  been 
surpassed  for  these  forms.  (2)  He  gave  the  first  correct 
account  of  the  maturation  divisions  and  origin  of  the  egg 
nucleus  (Hertwig  regarded  the  latter  as  being  the  per- 
sistent nucleolus  of  the  germinal  vesicle).  (3)  He  paid 
special  attention  to  the  origin  of  the  fertilization  mem- 
brane and  founded  the  classic  theory  that  it  was  an 
adaptation  to  prevent  polyspermy.  (4)  He  was  the 
first  one  adequately  to  present  the  harmful  effects  of 
polyspermy. 

The  period  initiated  by  these  two  men  was  charac- 
terized mainly  by  the  repeated  demonstration  of  pene- 
tration of  the  spermatozoon,  the  formation  of  a  nucleus 
from  the  sperm  head,  and  the  fusion  of  this  nucleus  with 
the  egg  nucleus.  It  was  also  gradually  demonstrated 
that  the  egg  nucleus  is    genetically  derived   from   the 


HISTORY  OF  THE  FERTILIZATION  PROBLEM     1 7 

germinal  vesicle  by  karyokinetic  divisions.  Thus  the 
genetic  continuity  of  the  germ  nuclei  with  nuclei  of 
preceding  cell  generations  was  estabhshed.  As  yet  the 
character  of  the  fusion  of  egg  and  sperm  nuclei  had 
hardly  been  raised,  for  the  chromosome  problems  and 
hypotheses  were  in  a  nascent  state.  Flemming's  dis- 
coveries concerning  chromosomes  and  their  reproduction 
in  karyokinesis  by  spKtting  date  only  from  1876-78. 

All  the  problems  of  cell  morphology  were  in  a  fine 
state  of  fermentation  during  this  time,  the  really  classic 
period  of  cell  morphology;  the  foundations  of  our  present 
knowledge  of  cell  division  were  being  laid;  before  the 
decade  1870-80  it  had  been  firmly  established  that  cells 
arise  only  by  division  from  pre-existing  cells;  but  two 
views  of  the  origin  of  nuclei  were  still  held,  one  that  of 
free  formation,  according  to  which  the  nuclei  of  daughter- 
cells  had  no  genetic  connection  with  the  nucleus  of  the 
mother-cell,  and  the  other  that  nuclei  arise  by  division 
from  a  preceding  nucleus.  Little  by  little  as  a  result 
of  numerous  investigations  "by  many  investigators,  both 
zoologists  and  botanists,  the  matter  cleared  up.  In 
1878  Flemming  was  able  to  outline  the  whole  scheme 
of  karyokinesis  substantially  as  we  now  understand  it. 

The  fundamental  biological  principle  of  genetic  con- 
tinuity was  foreshadowed  by  the  founders  of  the  cell 
doctrine  and  was  more  or  less  distinctly  foreseen  by  some 
of  their  contemporaries,  as  in  the  case  of  Lallemand. 
It  was  yet  more  clearly  expressed  in  Virchow's  famous 
aphorism,  omnis  cellula  e  cellula  (1856) ;  but  it  could  not 
become  an  established  guiding  principle  in  genetic 
research  until  the  entire  cell  cycle  of  the  individual  life- 
history  was  worked  out  in  broad  outline,  until  the  process 


1 8  PROBLEMS  OF  FERTILIZATION 

of  cell  division  was  accurately  ascertained  and  applied 
to  the  genealogy  of  the  germ  cells,  until  the  respective 
parts  of  ovum  and  spermatozoon  in  the  origin  of  the  new 
generation  were  understood,  nor  until  the  hoary  doctrine 
of  spontaneous  generation  was  banished  bodily  from  the 
field  of  biology.  These  were  all  accomplishments  of 
that  great  decade  in  biological  research  (1870-80), 
for  which  the  studies  of  the  preceding  thirty  years  had 
furnished  ample  preparation.  The  entire  superstructure 
of  modern  genetic  research  rests  upon  the  foundations 
then  laid. 

Professor  Mark's  paper  on  Limax  (1881)  is  a  point 
of  departure  between  the  fertilization  studies  of  the 
seventies  and  those  that  were  to  follow.  Professor 
Mark  observed  that  the  pronuclei  come  together  but 
do  not  fuse  to  form  a  first  cleavage  nucleus,  as  had  been 
described  for  other  animals.  ''  The  first  cleavage  nucleus 
does  not  have  a  morphological  existence."  The  pro- 
nuclei persist  after  the  appearance  of  the  cleavage 
centers;    their  membranes  then  gradually  disappear. 

In  1883  Van  Beneden  pubhshed  his  now  classic 
paper  on  Ascaris.  The  pronuclei  do  not  fuse;  both  are 
included  in  a  single  amphiaster;  each  produces  two 
chromosomes;  these  divide  and  their  halves  form  the 
daughter-nuclei.  In  the  nuclei  of  the  first  two  cells  there 
are  thus  equal  numbers  of  male  and  female  elements; 
and  there  are  reasons  to  believe  that  even  in  these  two 
nuclei  they  do  not  fuse;  it  is  probable  that  they  remain 
distinct  in  all  derivative  cells,  including  the  immature 
eggs  and  spermatogonia.  In  the  egg  the  chromatin 
is  composed  of  two  distinct  parts,  and  ''it  is  legitimate 
to  suppose  that  each  is  the  equivalent  of  a  male  and  a 


HISTORY  OF  THE  FERTILIZATION  PROBLEiM     19 

female  chromosome,  and  that  in  the  formation  of  the 
polar  globules  each  throws  out  the  male  chromatin  which 
it  contains." 

Van  Beneden  by  a  veritable  stroke  of  genius  thus 
anticipates  the  entire  chromosome  doctrine  of  the  present 
time,  even  though  certain  aspects  of  his  interpretation 
were  not  entirely  fortunate. 

With  the  estabhshment  of  the  nuclear  theory,  des- 
tined soon  to  be  elevated  into  the  doctrine  of  chromosome 
individuality,  a  certain  duahty  of  cell  life  was  recognized 
in  which  nucleus  and  cytoplasm,  however  interdependent, 
were  regarded  as  playing  specific  roles.  But  there 
was  no  logical  reason  for  stopping  at  duality,  and  the 
centrosome  soon  came  to  be  recognized  under  Van 
Beneden's  and  Boveri's  leadership  as  a  third  organ  of 
cell  life  reproducing  itself  by  division.  The  development 
of  this  idea  was  due,  not  only  to  studies  of  karyokinesis, 
but  also  to  the  series  of  fertilization  studies  which  began 
with  Boveri's  classic  papers  on  Ascaris  (1887-88).  In 
these  papers  Boveri  is  convinced  of  the  necessity  of 
making  ''the  sharpest  distinction  between  fertilization 
and  heredity,  i.e.,  between  the  question  how  egg  and 
spermatozoon  produce  a  cell  capable  of  division,  and  the 
question  how  these  cells  come  to  be  capable  of  reprodu- 
cing the  quaHties  of  the  parents  in  the  offspring";  this 
distinction  has  since  been  generally  recognized.  Boveri's 
solution  of  the  fertilization  problem  was  in  terms  of  the 
centrosome  hypothesis:  the  egg  is  devoid  of  the  organ 
of  cell  division,  the  centrosome;  capacity  for  division, 
hence  the  initiation  of  the  developmental  processes, 
is  restored  through  the  introduction  of  a  centrosome  into 
the  egg  by  the  spermatozoon. 


20  PROBLEMS  OF  FERTILIZATION 

This  conception  exerted  a  dominating  influence  on 
the  series  of  fertiUzation  studies  which  followed;  the 
questions  as  to  the  origin  of  the  sperm  aster  with  its 
contained  centrosome  in  the  egg,  and  as  to  the  genetic 
continuity  of  the  sperm  centrosome  with  the  centro- 
somes  of  the  cleavage  amphiaster,  were  energetically 
investigated  by  a  series  of  students  for  the  next  fifteen 
years  or  more,  and  similar  studies  have  continued  with 
less  energy  down  to  the  present  time.  Collectively 
these  publications  constitute  a  fairly  adequate  record 
of  the  morphology  of  the  fertilization  process  in  ani- 
mals, a  large  part  of  which  was  furnished  by  American 
students. 

The  morphological  analysis  of  fertilization  seems 
now  to  be  fairly  complete;  there  may  still  be  disturb- 
ances, such  as  recent  attempts  to  trace  mitochondria 
back  to  the  sperm,  which  seems  destined  to  share  the 
adverse  fate  of  the  similar  attempt  to  trace  the  centro- 
somes  to  the  sperm;  but  there  is  not  likely  to  be  any 
great  modification  of  the  existing  data,  which  seem  to 
me  to  demonstrate,  effectively  if  not  absolutely,  that 
the  sperm  head  contains  all  the  substances  necessary  for 
fertiUzation.  We  have  thus  attained  a  more  or  less 
definitive  solution  of  the  morphological  relations  of  egg 
and  spermatozoon  in  the  fertilization  process. 

The  cytologist  working  with  chromosomes  and  the 
geneticist  with  Mendelian  factors  have  traced  maternal 
and  paternal  elements  through  the  life-history  in  a 
very  satisfactory  manner,  so  that  we  are  beginning  to 
see  how  certain  strands  of  the  web  of  life  cross  the  gap 
of  successive  generations.  It  remains  for  the  biology 
of  the  future  to  elucidate  the  chemical  foundations  of 


HISTORY  OF  THE  FERTILIZATION  PROBLEM     21 

chromosome  behavior  and  to  identify  the  Mendelian 
factors  in  these  chemical  foundations. 

The  problems  of  the  immediate  reaction  of  the  repro- 
ductive elements  and  the  physiology  of  fertilization  are 
not  touched  by  this  morphological  analysis,  though  they 
had  been  present  in  the  minds  of  investigators  from  the 
beginning.  The  experimental  investigation  of  these 
problems  dates  from  Spallanzani,  as  we  have  seen,  but 
they  did  not  become  dominant  until  the  morphological 
problems  of  fertilization  were  in  an  advanced  stage  of 
solution.  They  constitute,  however,  the  more  imme- 
diate problems  of  fertilization,  considered  in  a  restricted 
sense. 

We  have  had  two  lines  of  attack  since  the  studies 
of  Oskar  and  Richard  Hertwig  pubhshed  in  1887  really 
initiated  the  modern  period  in  the  physiology  of  fertiliza- 
tion. The  one  is  a  direct  experimental  analysis  of  the 
fertilization  process  itself;  the  other  is  the  attempt  to 
imitate  the  action  of  the  spermatozoon  by  chemical  and 
physical  agencies— in  short,  the  studies  on  artificial 
parthenogenesis.  I  shall  not  attempt  in  the  present 
chapter  to  deal  with  the  latter,  which  constitute  one  of 
the  most  interesting  and  suggestive  chapters  in  modern 
biology,  beyond  attempting  to  define  their  relation  to 
the  problems  of  fertilization  proper. 

It  was  soon  found  in  the  course  of  studies  on  arti- 
ficial parthenogenesis  that  no  single  physical  or  chemical 
agency  sufiices  to  initiate  development  in  all  eggs,  and 
that  when  the  various  agencies  effective  in  all  the  success- 
ful experiments  are  assembled  they  constitute  a  fairly 
complete  Hst  of  agencies  to  which  protoplasm  in  general 
is  irritable.     The  idea  then  arose  that  the  common  factor 


2  2  PROBLEMS  OF  FERTILIZATION 

must  be  the  effective  one,  but  no  common  factor  has 
been  found,  or  can  be  found,  in  the  agents  themselves; 
the  only  common  factors  are  in  the  reproductive  cells. 
This  leaves  the  method  of  parthenogenesis  in  the  same 
position  as  the  method  of  analysis;  that  is,  in  the  position 
of  determining  what  are  the  changes  in  the  egg  itself 
that  initiate  development,  and  what  is  the  nature  of  their 
dependency  upon  the  external  agent  or  spermatozoon. 
The  answer  to  these  questions  cannot  proceed  exclusively 
from  parthenogenetic  studies,  though,  to  the  extent  that 
the  same  questions  are  involved,  parthenogenesis  and 
fertilization  studies  must  furnish  the  same  answer.  But 
there  are  obviously  fundamental  problems  of  fertilization 
that  cannot  be  touched  by  methods  of  artificial  partheno- 
genesis. 

The  conditions  to  be  fulfilled  in  fertilization  involve, 
not  only  penetration  of  the  spermatozoon,  or  some  part 
of  it,  into  the  egg,  but  also  reaction  between  the  two, 
which  is  evidenced  by  the  behavior  of  both  partners; 
for  it  is  possible  for  a  spermatozoon  to  penetrate  an  egg 
and  no  reaction  to  be  evidenced  in  the  behavior  of  either 
the  egg  or  sperm,  as  when  immature  eggs  are  penetrated 
by  mature  spermatozoa.  We  may  therefore  speak  of 
Si  fertilization  reaction  when  the  behavior  of  both  partners 
indicates  that  the  process  is  proceeding  normally. 
Fertilization  has  its  quantitative  aspect,  and  the  reaction 
may  be  complete  or  exhibit  varying  degrees  of  incom- 
pleteness. For  a  normal  fertilization  reaction  certain 
internal  conditions  of  the  partners  and  certain  external 
conditions  of  the  medium  must  be  realized.  The  study 
of  the  external  conditions  throws  light  upon  the  reaction, 
because  the  nature  of  the  internal  conditions  mav  be 


HISTORY  OF  THE  FERTILIZATION  PROBLEM     23 

inferred  from   the  necessary,  from   the  inhibiting,  and 
from  the  favoring  conditions  of  the  medium. 

The  fertihzation  reaction,  like  all  biological  reactions, 
requires  certain  conditions  of  the  environment,  such 
as  definite  range  of  temperature  and  chemical  compo- 
sition of  the  medium.  In  the  first  place,  if  these  are 
exceeded  in  either  direction  so  far  as  to  injure  the  cells 
the  fertilization  reaction  either  does  not  take  place  or  it. 
is  rendered  abnormal.  The  cause  of  the  failure,  or  the 
abnormality,  in  such  cases  lies  in  some  change  of  the 
internal  composition  of  one  or  the  other  of  the  germ 
cells.  The  classic  experiments  of  this  kind  are  those 
of  Oskar  and  Richard  Hertwig  pubhshed  in  1887. 
These  investigators  studied  the  effects  of  high  tempera- 
ture, of  various  injurious  chemical  reagents,  and  of 
mechanical  shock  on  the  germ  cells  separately  before 
fertilization,  and  on  the  process  of  fertilization  itself 
at  various  stages.  Many  exceedingly  interesting  obser- 
vations were  made,  and  problems  were  raised  that  were 
not  then  ripe  for  solution.  Other  experiments  of  a 
similar  kind  have  since  been  made,  but  their  considera- 
tion properly  belongs  to  the  problems  of  the  internal 
factors,  for  the  phenomena  observed  depend  upon  inter- 
nal changes  of  the  germ  cells. 

In  the  second  place,  there  may  be  modifications  of 
the  medium  which  do  not  directly  injure  the  germ  cells, 
but  which  inhibit  or  favor  the  fertilization  reaction. 
Examples  of  inhibiting  phenomena  are  found  in  Pro- 
fessor Loeb's  studies  of  the  relations  of  ions  to  the  ferti- 
lization reaction,  or  my  own  on  the  inhibiting  action 
of  blood  or  tissue  secretions  of  the  same  species  on 
fertilization.     The  most  striking  example  of  conditions 


24  PROBLEMS  OF  FERTILIZATION 

favoring  fertilization  is  the  action  of  alkalis  in  enabling 
interclass  hybridization,  discovered  by  Jacques  Loeb. 
Such  experiments  furnish  important  data  for  the  anal- 
ysis of  the  reaction,  but  it  is  obvious  that  their  inter- 
pretation must  depend  upon  internal  conditions  of  the 
fertilization  reaction. 

In  the  third  place,  the  membranes  of  the  egg  and  of 
the  spermatozoon  must  influence  the  occurrence,  rate, 
and  extent  of  the  fertilization  reaction  according  to 
the  degree  of  their  permeability  to  the  substances 
concerned;  the  egg  membrane  is  of  course  more  espe- 
cially concerned;  its  role  in  the  occurrence  of  partheno- 
genesis has  been  studied  especially  by  R.  S.  Lillie; 
and  I  have  found  in  the  case  of  the  starfish  egg  that  a 
resistant  egg  membrane  may  entirely  block  the  ferti- 
lization reaction,  though  the  block  may  be  removed  by 
agents  that  render  the  membrane  more  permeable. 

The  internal  conditions  of  the  fertilization  reaction 
may  be  grouped  under  three  heads:  (i)  maturity  of 
the  germ  cells;  (2)  irreversibility  of  the  reaction; 
(3)  specificity  of  the  reaction. 

I.  Maturity. — Concerning  conditions  of  maturity  of 
the  spermatozoon,  but  little  definitely  is  known,  except 
that  it  will  not  fertilize  before  its  differentiation  is 
complete.  Whether  the  cause  of  this  lies  entirely 
in  deficient  motility,  or  partly  also  in  incomplete 
chemical  differentiation,  we  do  not  know,  though  there 
are  some  reasons  for  thinking  that  the  latter  factor  may 
be  involved.  In  the  case  of  the  ovum  our  knowledge 
is  in  a  much  more  advanced  stage.  We  know  that  the 
fertilizable  condition,  which  represents  the  final  maturity 
of  the  ovum,  arises  rather  suddenly,  usually  lasts  but 


nmui  imiif 

VLCSktkCMm 


HISTORY  OF  THE  FERTILIZATION  PROBLEM     25 

a  short  time,  and  is  lost  as  an  immediate  consequence 
of  the  fertihzation  reaction,  (a)  That  the  fertiHzable 
condition  arises  suddenly  has  been  shown  especially 
by  the  work  of  Delage  on  the  starfish  egg  and  of  Wilson 
on  the  egg  of  Cerebratulus.  Their  experiments  on 
merogony  showed  that  parts  of  the  full-grown  ovum 
taken  prior  to  the  rupture  of  the  germinal  vesicle  are 
incapable  of  fertilization;  but,  soon  after  the  rupture  of 
the  germinal  vesicle,  parts,  whether  nucleated  or  not, 
readily  fertilize.  Hertwig's  observations  (1877)  also 
showed  a  complete  failure  of  the  fertilization  reaction 
in  primary  ovocytes  of  the  sea  urchin  before  rupture 
of  the  germinal  vesicle,  even  when  spermatozoa  pene- 
trated. I  have  observed  the  same  thing  in  Chaetop- 
terus.  (b)  Eggs  of  Platynereis  lose  their  capacity  for 
fertilization  ahnost  immediately  after  coming  into  sea- 
water,  even  though  spermatozoa  may  penetrate  (Just) ; 
eggs  of  the  frog  become  unfertilizable  after  half  an  hour 
in  water  (Spallanzani) ;  eggs  of  the  wall-eyed  pike  com- 
pletely lose  their  fertihzabiHty  after  ten  minutes  in 
water  (Reighard) .  Usually  fertilization  capacity  begins 
to  fall  off  in  one  or  two  hours  after  eggs  are  laid  in  niost 
marine  animals,  though  in  some,  as  in  the  sea  urchin, 
it  may  persist  much  longer. 

2.  Irreversibility. — Once  fertilized,  eggs  do  not  ferti- 
lize again,  nor  do  parts  of  such  eggs  that  are  freed  of 
the  fertilization  membrane.  It  should  therefore  be 
impossible  to  superimpose  parthenogenesis  and  ferti- 
lization; and  the  studies  of  C.  R.  Moore  show  this  to  be 
the  case.  Apparent  superposition  appears  in  all  cases 
to  be  due  to  incomplete  reactions,  which  cease  and 
may  be  subsequently  resumed.     The  fertilization  reaction 


26  PROBLEMS  OF  FERTILIZATION 

appears  to  be  irreversible ;  and  the  appearance  of  reversal 
in  parthenogenesis  may  be  referred,  like  superposition 
of  fertihzation  on  parthenogenesis,  to  incompleteness  of 
the  initial  reaction. 

3.  Specificity  is  an  outstanding  feature  of  the  ferti- 
hzation reaction,  the  significance  of  which  is  not  weak- 
ened by  any  hybridization  expermients.  We  need  not 
stop  to  define  the  limits  or  the  consequences  of  hybridi- 
zation in  order  to  justify  the  assertion  that  no  theory  of 
fertilization  which  fails  to  include  the  factor  of  specificity 
as  one  of  the  prime  elements  can  be  true. 

The  fundamental  character  of  specificity  is  illumi- 
nated by  the  phenomena  of  self-sterility;  in  species 
where  this  occurs  the  eggs  and  sperm  of  the  same  indi- 
vidual are  sterile  inter  se,  though  fertile  with  those  of 
all  other  individuals.  This  has  led  some  botanists  to 
the  conception  of  individual  stuffs;  but  Correns'  ex- 
perimental analysis  led  him  to  the  conclusion  that  the 
specific  factor  is  not  an  individual  stuff  but  a  definite 
combination  of  stuffs  for  each  individual.  The  combi- 
nation arises  always  with  the  individual  and  disappears 
with  it.  The  only  biological  parallel  of  such  phenomena 
is  found  in  the  individual  blood  composition  revealed  by 
serological  studies.  That  there  is  a  common  factor  in 
species  and  individual  specificity  no  one  who  has  studied 
both  sets  of  phenomena  can  doubt. 

A  consistent  theory  of  fertilization  must  take  account 
of  all  these  phenomena,  not  only  the  internal  factors  of 
maturity  of  germ  cells  and  the  specificity  of  their  reac- 
tions, but  also  the  external  factors  that  favor  or  inhibit 
the  reaction.  I  have  attempted  to  show  in  a  series  of 
papers  that  the  fertilizable  condition  of  the  egg  depends 


HISTORY  OF  THE  FERTILIZATION  PROBLEM     27 

upon  the  presence  of  a  specific  substance  which  is  pro- 
duced at  the  time  of  rupture  of  the  germinal  vesicle  and 
which  disappears  completely  after  fertilization.  If  this 
substance  be  present  in  the  egg  in  adequate  amount  the 
egg  can  be  fertilized,  otherwise  not.  It  may  be  obtained 
in  solution  in  the  sea-water  and  recognized  by  its  capacity 
for  agglutinating  sperm  suspensions  of  the  same  species, 
in  some  cases  at  least.  If  it  is  thus  possible  to  associate 
the  fertilizable  condition  of  the  ovum  with  a  definite 
substance,  we  have  a  base  from  which  an  analysis  of 
fertilization  can  be  made. 

If  the  existence  of  such  a  substance  be  admitted, 
it  must  operate  either  by  activating  some  substance  in 
the  spermatozoon,  which  is  to  be  regarded  as  the  effective 
agent  in  subsequent  changes,  or  we  must  regard  it  as  the 
effective  agent  which  is  transformed  from  an  inactive  to 
an  active  state  by  some  substance  in  the  spermatozoon. 
If  we  take  the  first  alternative,  we  have  no  explanation  of 
parthenogenesis,  whereas  if  we  regard  the  egg  substance 
as  the  active  agent,  the  explanation  of  parthenogenesis 
proceeds  along  the  same  lines  as  that  of  fertihzation. 
Moreover,  I  have  been  able  to  show  by  an  analysis  of  the 
phenomenon  of  blood  inhibition  of  fertilization  that  the 
first  point  of  view  is  untenable. 

This  substance  may  therefore  be  called  the  fertiUzing 
substance,  or  fertilizin.  By  its  reaction  it  is  shown  to 
be  a  colloidal  substance,  not  giving  the  usual  protein 
tests,  and  exhibiting  some  of  the  properties  of  a  ferment, 
as  shown  by  Richards  and  Woodward.  Fertilization 
would  thus  be  a  three-body  reaction  between  the  sperm 
receptors,  fertilizin,  and  egg  receptors  Hnked  in  line; 
and  it  is  possible  to  show  that  inhibiting  agencies  may 


28  PROBLEMS  OF  FERTILIZATION 

operate  at  the  various  linkages  of  such  a  reaction.  In 
its  reaction  with  the  sperm  the  fertilizin  of  different 
species  exhibits  a  certain  degree  of  specificity,  which 
should  be  more  fully  studied,  but  which  has  been  partly 
explored  by  Jacques  Loeb  and  myself. 

This  form  of  hypothesis  takes  into  account  the  inter- 
nal factors  both  of  maturity  of  germ  cells  and  of  their 
specificity;  it  is  also  adapted  to  explain  the  environ- 
mental conditions  of  fertilization  extremely  well;  and 
it  is  consistent  with  the  results  of  parthenogenesis,  and 
the  known  relations  of  parthenogenesis  and  fertilization 
to  the  permeable  or  impermeable  conditions  of  the  egg 
membrane. 

In  the  following  pages  we  shall  discuss  the  various 
problems  whose  history  has  thus  been  outlined.  The 
discussion  can  be  no  more  than  a  record  of  progress. 

REFERENCES 

AuERBACH,  Leopold. 

1874.     Organologische  Studien  (see  pp.  1^^-262).     Breslau. 

BlSCHOFF,  T.  L.  W. 

1842.     Entwickelungsgeschichte  des  Kaninchens  (chap.  ii). 
1847.     "Theorie  der  Befruchtung  und  liber  die  Rolle,  welche 

die  Spermatozoiden  dabei  spielen,"  Arch,  fiir  Anat. 

u.  Phys.,  pp.  422-42. 

B  OVERT,   T. 

1887.  "Ueber  die  Befruchtung  der  Eier  von  Ascaris  megalo- 
cephala,"  Sitzimgsher.  d.  Gesell.  fiir  Morph.  u.  Phys. 
(Miinchen),  Band  3,  pp.  71-80. 

1887.  "Ueber  den  Antheil  des  Spermatozoons  an  der 
Theilung  des  Eies,"  ibid.,  pp.  154-64. 

1887.  "Zellenstudien,"  Heft  I,  Jen.  Zeitschr.  fur  Naturw., 
Band  21,  pp.  423-51 S- 

1888.  "ZeUenstudien,"  Heft  II,  ibid.,  Ba.nd  22, pp.  685-882. 


HISTORY  OF  THE  FERTILIZATION  PROBLEM     29 

1890.  "ZeUenstudien,"  Heft  III,  ibid.,  Ba.nd  24,  pp.  314-401. 

1891.  "Befruchtung,"  Ergeb.  der  Anal,  und  Entwicke- 
lungs geschichle,  Band  i,  pp.  364-485. 

BUTSCHLI,    O. 

1875.  "  Vorlaufige  Mittheilung  iiber  Untersuchungen  betref- 
fend  die  ersten  Entwickelungsvorgiinge  im  befruch- 
teten  Ei  von  Nematoden  und  Schnecken,"  Zeitschr. 
fur  wiss.  ZooL,  Band  25,  pp.  201-16. 

1876.  "Studien  iiber  die  ersten  Entwickelungsvorgange  der 
Eizelle,  die  Zelltheilung  und  die  Conjugation  der 
Infusorien,"  Abh.  d.  Senck.  Naturf.  Gesell.,  Band 
10,  pp.  213-464. 

F6l,  Hermann. 

1876.  "Sur  les  phenomenes  intimes  de  la  division  ccllu- 
laire,"  Comptes  rendus  de  VAcad.  des  Sci.,  T.  87,,  pp. 
667-69. 

1877.  "Sur  le  commencement  de  I'henogenie  chez  divers 
animaux,"  Arch,  de  zool.  exp.  et  gen-.,  T.  6,  pp.  145-69. 

1879.  ''Recherches  sur  la  fecondation  et  le  commencement 
de  I'henogenie  chez  divers  animaux,"  Geneve  soc. 
phys.  mem.,  XXVI,  89-397. 

Hartsoeker,  Nicholas. 

1694.     Essay  de  dioptrigue  (pp.  22j-T,2,).     Paris. 

Harvey,  William. 

1 65 1.     De  generatione  animalium. 

Hertwig,  O. 

1876.  "Beitriige  zur  Kenntniss  der  Bildung,  Befruchtung 
und  Theilung  des  thierischcn  Eies,"  Morph.  Jahrb., 
Band  I,  pp.  347-434- 

1877.  Theil  II,  ibid.,  Band  III,  pp.  1-86. 

1878.  Theil  III,  ibid.,  Band  IV,  pp.  156-213. 

Hertwig,  O.  and  R. 

1887.  "Ueber  die  Befruchtungs-  und  Theilungsvorgiinge 
des  thierischen  Eies  unter  den  Einfluss  iiusserer 
Agentien,"  Jen.  Zeitschr.  Jiir  Naturd.'.,  Band  20, 
pp.  120-241,  477-510. 


so  PROBLEMS  OF  FERTILIZATION 

Lallemand 

1 84 1.  ''Observations  sur  le  role  des  zoospermes  dans  le  gene- 
ration," Ann.  des  sci.  nat.,  Ser.  2,  T.  15,  pp.  262-307. 
Leeuwenhoek,  Antonius. 

1677.    Phil.  Trans.,  XI-XII,  1040-43. 

1699.     Ibid.,  XXI,  301-8. 

Mark,  E.  L. 

1881.  "Maturation,  Fecundation  and  Segmentation  of 
Limax  campestris,"  Bull.  Miis.  Comp.  Zool.  Harvard 
University,  VI,  173-625. 

Newport,  George. 

1854.  "On  the  Impregnation  of  the  Ovum  in  the  Amphibia," 
First  Series,  Phil.  Trans.,  CXLI,  169-242;  Third 
Series,  ibid.,  CXLIV,  229-44. 

Prevost  et  Dumas. 

1824.     "Nouvelle    theorie    de    la    generation,"    Ann.    des 

sci.  nat.,  T.   i,  pp.  1-29,    167-87,    274-92;    T.  11, 

pp.  100-121,  129-49. 
1827.     "Memoir    sur    le    developpement    du    poulet    dans 

I'oeuf,"  ibid.,  T.  12,  pp.  414-43. 

Spallanzani,  Abbe. 

1785.    Experiences  pour  servir  a  I'histoire  de  la  generation 
des  animaux  et  des  plantes.     Geneva. 
Van  Beneden,  E. 

1875.  "La  maturation  de  I'oeuf,  la  fecondation  et  les  prem- 
ieres phases  du  developpement  embryonaire  des 
mammiferes,"  etc..  Bull,  de  I' Acad.  Roy.  de  Belgique, 
Ser.  II,  T.  40,  pp.  686-736.  See  also  Qiiart.  Jour. 
Micr.  Sci.,  XVI  (1876),  153-82. 

1883.  "Recherches  sur  la  maturation  de  I'oeuf  et  la  feconda- 
tion," Arch,  de  blol.,  T.  4,  pp.  265-640. 


CHAPTER  II 
THE  PLACE  OF  FERTILIZATION  IN  THE  LIFE-HISTORY 

Fertilization  is  essentially  the  phenomenon  of  the 
union  of  two  cells  known  as  gametes  to  form  a  single 
cell  known  as  the  zygote.  Considered  in  this  broad 
sense,  it  is  practically  a  universal  phenomenon  among 
animals  and  plants.  The  formation  and  union  of  gametes 
probably  always  goes  hand  in  hand  with  sex,  even  in 
those  cases  among  the  Protista  when  the  gametes  have 
not  yet  been  shown  to  be  sexually  differentiated. 

There  is  perhaps  no  phenomenon  in  the  field  of 
biology  that  touches  so  many  fundamental  questions 
as  the  union  of  the  germ  cells  in  the  act  of  fertilization; 
in  this  supreme  event  all  the  strands  of  the  webs  of  two 
lives  are  gathered  in  one  knot,  from  which  they  diverge 
again  and  are  re-woven  in  a  new  individual  life-history. 
It  is  the  central  decisive  event  in  the  genesis  of  all  sexually 
produced  animals  and  plants.  Thus  from  one  point  of 
view  it  envisages  the  entire  problem  of  sex;  from  another 
point  of  view  it  constitutes  the  basis  of  all  development 
and  inheritance.  The  elements  that  unite  are  single 
cells,  each  usually  incapable,  under  natural  conditions, 
of  continued  existence  or  development — on  the  point  of 
death;  but  by  their  union  a  rejuvenated  individual  is 
formed  which  constitutes  a  link  in  the  eternal  procession 
of  life  by  virtue  of  its  power  of  reproduction.  Thus  to 
consider  the  antecedents  and  the  consequents  of  the 
process  of  fertilization  would  be  to  outline  all  of  biology. 

31 


32  PROBLEMS  OF  FERTILIZATION 

Our  purpose,  however,  is  to  focus  on  the  moment  of 
fertihzatioji  itself,  feeling  sure  of  the  great  significance 
of  any  adequate  analysis  of  such  an  event.  Even  so 
doing,  we  are  dealing  with  a  highly  complex  situation 
in  which  all  of  our  knowledge  of  the  morphology  and 
physiology  of  cells  is  requisite  for  a  statement  of  the 
problems. 

The  biologist  who  surveys  the  stupendous  panorama 
of  sex  in  the  plant  and  in  the  animal  kingdom  must 
feel  that  there  is  a  universal  biological  significance  of 
fertilization,  in  terms  of  either  function  or  composition. 
By  some  biologists  emphasis  has  been  laid  on  the  aspect 
of  rejuvenation  and  by  others  on  the  mingling  of  the 
parental  germ  plasms  as  a  source  of  diversity  and  vari- 
ation within  the  species — on  amphimixis,  in  brief. 

The  development  of  an  organism  after  a  very  early 
stage  is  characterized  by  decreasing  rate  of  metabolism; 
that  is  to  say,  at  a  certain  early  stage,  which  will  be  more 
•fully  defined  later,  the  organism  possesses  its  maximum 
growth  energy,  which,  in  the  case  of  a  mammal  for 
instance,  may  be  more  than  a  hundred  times  that 
possessed  at  birth.  The  growth  energy  decreases  from 
its  maximum  point  most  rapidly  at  first,  with  in- 
creasingly diminishing  rate  until  full  growth  is  attained. 
Thereafter  the  processes  of  growth  and  destruction  prac- 
tically balance,  for  a  time,  up  to  old  age  and  death. 

Charles  S.  Minot  deserves  the  credit  for  first  clearly 
defining  these  principles  in  a  quantitative  way.  He 
showed  that  in  the  early  stages  of  embryonic  develop- 
ment, from  the  ninth  to  the  fifteenth  day  of  gestation, 
rabbits  add  704  per  cent  to  their  weight  daily,  and 
inferred    that    in   yet   earlier   stages    the   rate   of   in- 


FERTILIZATION  IN  THE  LIFE-HISTORY  S3 

crement  was  probably  at  least  i,ooo  per  cent  daily. 
From  the  fifteenth  to  the  twentieth  day  of  gestation 
the  rabbit  adds  only  212  per  cent  daily  to  its  weight. 
Four  days  after  birth  the  increment  is  about  17  per 
cent  daily;  twenty- three  days  after  birth  onl}'  about 
6  per  cent;  two  months  after  birth  less  than  2  per 
cent;  at  two  and  one-half  months  less  than  i  per 
cent;  and  after  full  growth  is  attained,  about  two 
hundred  and  twenty  days  after  birth,  the  weight  no 
longer  increases  regularly.  The  processes  of  breaking 
down  and  repair  in  the  organism  tend  to  balance.  He 
showed  that  similar  principles  hold  in  the  development 
of  man,  guinea-pig,  and  hen. 

We  have  here,  indeed,  a  general  principle  of  develop- 
ment, which  presumably  holds  for  all  metazoa,  which 
may  be  expressed  by  saying  that  in  embryonic  stages 
the  period  of  most  rapid  loss  of  growth  power  occurs, 
and,  if  this  is  identical  with  the  process  of  growing  old, 
that  senescence  is  most  rapid  in  exceedingly  early  stages 
of  development.  The  underlying  conception  has  long 
been  appreciated  by  naturalists;  it  is  an  old  idea,  which 
it  would  be  difhcult  to  trace  to  its  ultimate  source,  that 
organisms  start  out  with  a  high  charge  of  growth  energy, 
which  is  gradually  dissipated  in  the  process  of  growing 
old. 

The  point  of  maximum  efhciency  of  the  organism  in 
the  life-history  does  not,  however,  correspond  with  the 
starting-point,  the  fertilized  egg,  but  to  an  early  follow- 
ing stage  of  development.  The  germ  cells  themselves 
have,  indeed,  in  the  process  of  their  differentiation 
completely  lost  growth  capacity,  and  exhibit  also  in 
terms  of  differentiation  all  the  signs  of  senescent  cells. 


34  PROBLEMS  OF  FERTILIZATION 

Following  the  union  of  the  ovum  and  spermatozoon 
in  the  act  of  fertilization,  there  is  a  rapid  acceleration  of 
metabolism,  which  can  be  measured  in  various  ways, 
mounting,  in  stages  succeeding  cleavage,  to  a  maximum 
from  which  the  curve  of  senescence,  which  has  been 
outlined  above,  takes  its  start. 

Fertilization  thus  initiates  a  period  of  rejuvenescence 
which  is  brief  and  rapid  and  apparently  complete. 
However,  in  forms  that  reproduce  asexually,  rejuvenes- 
cence may  be  effected  in  other  ways  without  any  partici- 
pation of  gametes;  and  in  parthenogenesis  a  certain 
amount  of  rejuvenescence  at  least  occurs  without  ferti- 
lization. In  most  such  animals,  however,  sexual  repro- 
duction and  fertilization  occur  either  regularly  or 
irregularly  in  certain  generations. 

It  is  thus  readily  seen  how  the  idea  of  rejuvenescence 
came  to  be  associated  with  the  fertilization  problem. 
The  germ  cells  alone  in  most  animals  (and  all  the  higher 
ones)  possess  this  capacity  of  resuming  youthful  con- 
ditions after  differentiation,  and  they  do  so  (except  in 
case  of  parthenogenesis)  only  after  union  in  fertilization. 
The  exceptions,  however,  show  that  in  this  respect 
fertilization  is  not  an  absolutely  unique  process. 

Child  (191 5,  p.  58)  defines  senescence  and  rejuvenes- 
cence as  follows:  ''Senescence  is  primarily  a  decrease 
in  rate  of  dynamic  processes  conditioned  by  the  accumu- 
lation, differentiation,  and  other  associated  changes  of 
the  material  of  the  colloid  substratum.  Rejuvenescence 
is  an  increase  in  rate  of  dynamic  processes  conditioned 
by  the  changes  in  the  colloid  substratum  in  reduction 
and  dedifferentiation. "  If  we  accept  this  definition  as  a 
reasonable  physiological  formulation  of  the  processes 


FERTILIZATION  IN  THE  LIFE-HISTORY  35 

of  senescence  and  rejuvenescence,  we  gain  a  physiological 
conception  of  the  sense  in  which  the  mature  gametes 
may  be  termed  old  or  senescent,  and  of  the  sense  in  which 
fertilization  may  be  said  to  induce  a  process  of  rejuvenes- 
cence. 

The  gametes  are  old  in  the  sense  of  possessing 
a  high  degree  of  differentiation.  The  egg  cell  is  more  or 
less  loaded  with  a  highly  inert  material,  the  yolk,  and  the 
spermatozoon  is  highly  differentiated  in  a  different  sense. 
With  the  activation  of  the  ovum  by  fertilization  there 
begins  a  process  of  resorption  and  utilization  of  yolk, 
and  the  cleavage  cells  gradually  consume,  and  thus 
free  themselves  from,  this  encumbrance.  The  differen- 
tiated parts  of  the  spermatozoon  are  lost,  and  the  nucleus 
enters  into  the  nucleoplasmic  activities  of  the  zygote. 
It  is  improbable  that  this  is  the  whole  story  of  dedif- 
ferentiation,  and  Child  lays  strong  emphasis  in  this 
definition  and  elsewhere  on  changes  in  the  colloidal 
system,  but  the  nature  of  such  changes  in  relation 
to  the  process  of  rejuvenescence  remains  unknown. 

The  inner  significance  of  sex  and  fertilization  in  the 
life-history  cannot,  however,  be  confined  within  these 
boundaries.  Sex  seems  to  be  very  nearly  a  universal 
attribute  of  living  beings,  either  actual  or  potential, 
even  in  organisms  that  do  not  utilize  it  invariably  for 
purposes  of  reproduction;  and  it  hardly  suffices  to  say 
that  in  such  cases  the  condition  is  an  inherited  one  that 
has  lost  partially  or  wholly  its  function  in  the  life- 
history.  Sexual  differentiation  seems  to  represent  an 
inevitable  dimorphism  of  living  matter  for  which  we 
possess  no  satisfactory  analogy,  and  fertilization  appears 
to  be  a  consequence  of  such  dimorphism.    The  gametes 


36  PROBLET^IS  OF  FERTILIZATION 

are  not  merely  senescent,  if  we  take  this  view  of  their 
differentiation,  but  they  possess  properties  with  re- 
spect to  one  another  that  no  other  senescent  cells 
possess  with  reference  either  to  one  another  or  to  the 
gametes. 

The  second  most  general  aspect  of  fertilization  con- 
cerns the  union  in  one  individual  of  materials  derived 
from  two,  and  the  consequent  combination  of  certain 
characters  of  each  of  the  parents  in  the  zygote.  The 
entire  process  of  biparental  inheritance  depends  on  the 
preparation  of  the  materials  prior  to  union,  the  nature 
of  the  union  itself,  and  the  subsequent  redistribution  of 
such  materials  in  development.  Since  fertihzation  deals 
only  with  the  nature  of  the  union,  we  shall  not  be  con- 
cerned with  processes  of  inheritance.  It  will  therefore 
sufhce  to  point  out  here  the  most  general  impHcations 
of  this  aspect  of  the  subject. 

The  union  is  undoubtedly  the  chief  source  of  the 
heritable  variations  that  we  can  directly  observe.  In 
a  freely  interbreeding  population  the  germ  plasms  of 
no  two  individuals  are  precisely  alike,  and  the  differences 
often  extend  to  numerous  factors.  The  combinations 
and  recombinations  that  occur  as  a  result  of  fertihzation 
in  successive  generations  produce,  therefore,  the  greatest 
possible  amount  of  diversity  of  which  the  population  is 
capable  on  the  basis  of  the  organization  of  the  germ 
plasm,  assuming  equal  chances  and  viability  of  all 
crosses.  Material  is  thus  offered  for  natural  selection 
to  work  on.  But  beyond  this  there  is  a  relation  between 
vigor  of  offspring  and  zygotic  combinations,  in  very 
many  cases  at  least,  in  favor  of  heterozygous  as  compared 
with    homozygous    matings.     Certainly    not    all    com- 


FERTILIZATION  IN  THE  LIFE-HISTORY  37 

binations  produce  equal  vigor,  and  one  factor  in  many 
cases  in  the  production  of  increased  vigor  of  offspring  is 
found  in  the  union  of  contrasting  germ  plasms,  whether 
with  reference  to  one  or  to  several  characters. 

These  several  problems  appear  in  their  simplest 
forms  in  the  life-histories  of  certain  Protozoa,  and  they 
have  been  studied  most  intensively  in  ciliates,  especially 
in  Parameciiim.  It  has  long  been  recognized  that  for  a 
true  comparison  of  the  protozoan  life-cycle  to  that  of 
Metazoa,  the  period  between  two  divisions  does  not 
suffice.  It  is  true  that  two  individuals  result  from  such 
a  division,  but  these  are  comparable  in  many  respects 
to  cell  generations  in  the  metazoan  body,  with  the  main 
difference  that  the  cells  remain  associated  in  the  latter 
case  but  not  in  the  former. 

Looked  at  in  this  way,  it  is  frequently  possible  to 
distinguish  periods  of  youth,  maturity,  and  senescence 
in  a  protozoan  life-history  comparable  to  the  same 
period  of  a  metazoan  individual.  This  point  of  view 
goes  back  to  Butschli  (1876),  but  was  first  placed  on  an 
experimental  basis  by  Maupas  (1888-89),  who  regarded 
the  process  of  conjugation  in  ciHates  as  a  rejuvenating 
process:  The  ex-conjugant  begins  a  cycle  of  cell  gener- 
ations with  a  full  store  of  energy;  under  suitable  nutritive 
conditions  a  series  of  fissions  follows.  During  the  early 
part  of  the  series  there  is  no  regular  tendency  toward 
conjugation,  but  later  in  the  series  (after  several  hun- 
dred generations  usually)  the  tendency  to  conjugate  in 
pairs  becomes  very  strong  and  may  result  in  veritable 
epidemics  of  conjugation.  Maupas  calls  this  the  stage 
of  sexual  maturity.  Individuals  that  do  not  succeed 
in  conjugating  then   gradually   pass  into   a  period   of 


SS  PROBLEMS  OF  FERTILIZATION 

senescence  ending  in  death.  The  ex-conjugants  are, 
however,  rejuvenated  and  begin  new  Hfe-cycles. 

Maupas  thus  distinguished  periods  of  youth,  sexual 
maturity,  and  old  age  in  the  ciliate  life-cycle;  and  he 
believed  that  conjugation  brought  about  a  complete 
renewal  of  youth.  He  beheved,  moreover,  that  diverse 
ancestry  of  the  conjugating  individuals  was  important 
for  rejuvenation,  and  that  union  of  too  closely  related 
individuals  was  likely  to  be  abortive  in  this  respect. 

These  views  stood  in  the  sharpest  contrast  to  those 
of  Weismann,  who  held  ''that  the  deeper  significance 
of  every  form  of  amphimixis — whether  occurring  in 
conjugation,  fertihzation,  or  in  any  other  way — consists 
in  the  creation  of  that  hereditary  individual  variability 
which  is  requisite  for  the  operation  of  the  process  of 
selection,  and  which  arises  from  the  periodical  minghng 
of  the  individually  different  hereditary  substances." 
Moreover,  Weismann  was  not  simply  content  to  empha- 
size this  aspect  of  conjugation,  but  he  also  rejected  and 
ridiculed  the  rejuvenation  conception:  "To  my  mind  it 
is  difficult  to  understand  how  an  almost  exhausted  vital 
force  could  be  raised  again  to  its  original  state  of  activity 
as  the  consequence  of  a  union  with  another  equally 
exhausted  force."  Of  course  such  a  proposition  gains 
its  strength  from  its  form  rather  than  from  its  sub- 
stance. 

To  explain  the  death  of  individuals  that  fail  to  con- 
jugate, Weismann  supposes  that,  when  the  long  prepared 
period  of  conjugation  approaches,  the  appropriate  nuclear 
maturation  processes  occur;  the  result  of  such  matura- 
tion is  to  organize  the  individuals  as  gametes,  which  die 
like  any  gametes  which  fail  to  unite. 


FERTILIZATION  IN  THE  LIFE-HISTORY  39 

These  problems  have  been  studied  very  intensely 
in  Paramecium  in  recent  years  by  Calkins,  Woodruff, 
Woodruff  and  Erdmann,  Jennings,  and  others.  Most 
observers  have  detected  cycles  in  the  life-history 
consisting  of  ''a  more  or  less  periodic  alternation  of 
high  and  low  vitahty  as  measured  by  the  division  rate" 
(Calkins,  191 5).  The  period  of  depression  or  low  vitality 
may  lead  to  conjugation  in  certain  strains  exactly  as 
Maupas  observed;  on  the  other  hand,  high  vitality  may 
be  restored  by  certain  changes  in  the  culture  medium,  as 
Calkins  showed;  or  high  vitality  may  be  resumed  appar- 
ently spontaneously  without  conjugation  over  and  over 
again  for  periods  of  years  and  through  thousands  of 
generations,  as  Woodruff  first  observed  in  a  certain 
strain  of  Paramecium  aurelia.  The  analysis  of  the  last 
case  showed  that  each  such  period  of  depression  and 
restoration  was  accompanied  by  a  process  of  nuclear 
reorganization  comparable  in  many  respects  to  those 
taking  place  in  conjugation:  the  macronucleus  breaks 
up  and  is  finally  resorbed,  the  micronuclei  divide  twice, 
but  do  not  carry  out  the  third  division,  which  in  conjuga- 
tion produces  the  gametic  nuclei;  a  new  macronucleus 
is  formed  from  the  micronuclei,  and  the  normal  nuclear 
organization  is  gradually  restored. 

To  this  spontaneously  recurring  process  of  reorgani- 
zation Woodruff  has  applied  the  name  ''endomixis," 
thus  emphasizing  its  resemblance  in  many  respects 
to  amphimixis.  He  finds  that  this  process  is  essential  to 
the  continuation  of  the  life  of  the  culture  (Woodruft*, 
191 7),  for  its  discontinuance  is  invariably  followed, 
within  the  time  of  one  or  two  rhythms,  by  death  of  the 
the    culture.     Moreover    its    regular   periodicity    is    a 


40  PROBLEMS  OF  FERTILIZATION 

function  of  time  rather  than  of  cell  generations.  It 
is  obviously  an  expression  of  senescence  in  its  descending 
phase  and  of  rejuvenescence  in  its  ascending  phase 
if  these  terms  are  to  have  any  precise  biological  signifi- 
cance. These  organisms  may  thus  continue  to  live 
indefinitely,  as  Weismann  contended,  but  they  are  not 
eternally  young,  though  they  bear  with  them  a  foun- 
tain of  youth  in  the  process  of  endomixis  that  may  cause 
indefinite  rejuvenation. 

How,  now,  does  this  determination  bear  on  the  ques- 
tion of  whether  conjugation  does  or  does  not  involve 
a  rejuvenation  process?  Obviously  it  involves  no 
contradiction,  for  in  conjugation  we  have  an  equally 
complete  process  of  nuclear  reorganization;  the  added 
factor  of  nuclear  exchange  between  the  partners  and 
and  nuclear  syngamy  in  each  may,  however,  act  detri- 
mentally to  the  life  of  the  organism,  as  Jennings  showed. 
Endomixis  definitely  demonstrates,  however,  that  the 
rejuvenescence  theory  of  conjugation  went  too  far  in 
asserting  that  conjugation  itself,  presumably  in  all  its 
phases,  is  necessary  for  rejuvenescence.  Calkins  has 
interpreted  the  phenomenon  of  endomixis  as  a  kind  of 
parthenogenesis,  admitting  its  rejuvenating  influence; 
but  in  so  doing,  it  seems  to  the  writer,  he  has  relinquished 
the  most  distinctive  part  of  Maupas'  theory.  Both 
Calkins  and  Jennings,  moreover,  have  shown  that 
conjugation  of  even  the  most  closely  related  individuals 
has  no  injurious  effect,  and  have  thus  removed  another 
pillar  of  Maupas'  edifice. 

Jennings  (191 2)  has  also  ingeniously  shown  that  if 
conjugants  are  separated  in  the  very  first  stage  of  the 
process  they  may  continue  to  multiply  with  undimin- 


FERTILIZATION  IN  THE  LIFE-HISTORY  41 

ished  vigor,  and  he  points  out  that  we  cannot  therefore 
assume,  ''as  has  been  done  by  some  authors,  that  if  the 
animals  continue  to  multiply  after  conjugation,  this 
shows  that  conjugation  has  had  a  rejuvenating  effect, 
for  the  same  specimens  continue  equally  without  con- 
jugation." The  phenomenon  of  endomixis  was  not 
known  at  the  time  that  this  was  written,  and  Jennings 
therefore  did  not  determine  its  occurrence  in  the  sepa- 
rated conjugants;  but  it  is  reasonable  to  assume  that  it 
occurred  soon,  if  not  immediately,  because  it  is  apparently 
a  normal  process  in  all  races  of  Paramecium.  He  con- 
cludes from  his  very  extensive,  well-controlled  series 
of  experiments  that  "there  is  no  evidence  that  conjuga- 
tion in  the  infusoria  increases  the  reproductive  power, 
or  rejuvenates  the  organism  physiologically  in  any  way" 
(Jennings,  1913a). 

In  these  experiments  Jennings  demonstrated  that 
conjugation,  on  the  average,  decreases  the  rate  of  fission 
very  greatly  instead  of  increasing  it;  it  produces  a 
great  amount  of  variation  in  this  respect,  ranging  from 
a  total  loss  of  capacity  for  fission  to  a  rate  nearly  equal 
to  the  original,  and  also  variations  in  other  character- 
istics. "What  conjugation  does  is  to  bring  about  new 
combinations  of  germ  plasm,  just  as  is  done  in  the  sexual 
reproduction  of  higher  animals.  One  result  of  this  is 
to  give  origin  to  many  variations,  in  the  sense  of  inher- 
ited differentiations  between  different  strains.  Some  of 
the  new  combinations  are  better  adapted  to  the  existing 
conditions  than  others;  these  survive  while  the  others 
die  out"  (Jennings,  1913^). 

In  respect  to  conjugation,  Paramecium  and  the  cili- 
ates  generally  are  quite  aberrant  in  comparison  with  other 


42  PROBLEMS  OF  FERTILIZATION 

protozoan  groups.  Other  types  of  Protozoa  exhibit 
frequently  a  more  definite  type  of  Hfe-cycle,  ''so  that  a 
cell  picked  out  at  one  phase  of  the  hfe-cycle  is  quite  a 
different  type  of  individual  from  one  picked  out 
at  another  phase"  (Calkins,  191 6).  In  these  cases, 
conjugation  occupies  a  definite  place  in  the  hfe-cycle 
quite  comparable  to  its  place  in  Metazoa;  and  it  may 
apparently  be  equally  connected  with  processes  of  reju- 
venescence. However,  it  is  clear  that  rejuvenescence 
considered  as  a  process  of  differentiation  and  relief  from 
''cumulative  metabohc  differentiations"  (Calkins)  may 
be  accomplished  in  Protozoa,  as  in  certain  Metazoa,  at 
other  times  than  that  of  conjugation  (fertilization). 

The  only  result  of  conjugation  or  fertilization  common 
to  the  animal  kingdom  as  a  whole  is  biparental  inheri- 
tance. The  association  of  fertilization  with  repro- 
duction or  with  rejuvenescence  is  not  a  universal  one, 
and  therefore  not  a  necessary  one  in  the  most  general 
sense.  In  the  evolution  of  the  animal  kingdom,  however, 
the  processes  have  become  more  and  more  inseparably 
associated  in  the  higher  phyla,  so  that  sexual  repro- 
duction becomes  the  only  method  for  the  entire  organism, 
whether  of  reproduction  or  of  rejuvenescence. 

Sex  and  fertihzation  remain  for  the  present  ultimate 
biological  categories.  We  possess  innumerable  data 
concerning  their  manifestations  from  low  to  high  forms 
in  the  animal  kingdom;  and  the  researches  of  recent 
years  have  contributed  greatly  to  our  understanding  of 
the  physiology  and  genetics  of  sex,  and  the  morphology 
and  physiology  of  fertilization.  It  is  only  by  a  con- 
tinuation of  such  studies  that  we  can  hope  to  advance 
farther  into  the  problem  of  their  ultimate  significance. 


FERTILIZATION  IN  THE  LIFE-HISTORY  43 

REFERENCES 
Calkins,  Gary  N. 

191 5.  "Cycles  and  Rhythms  and  the  Problem  of  'Immor- 
tahty'  in  Parameciimi/'  Anicr.  Naturalist,  XLIX, 
65-76. 

1916.  "General  Biology  of  the  Protozoan  Life  Cycle," 
Amer.  Naturalist,  L,  257-70. 

Child,  CM. 

191 5.     Senescence  and  Rejuvenescence,  pp.  481.     The  Univer- 
sity of  Chicago  Press. 

Jennings,  H.  S. 

191 2.  "Age,  Death,  and  Conjugation  in  the  Light  of  Work 
on  Lower  Organisms,"  Pop.  Sci.  Monthly,  June, 
1912. 

1913.  "The  Effect  of  Conjugation  in  Paramecium,^'  Jour. 
Exp.  ZooL,  XIV,  279-391. 

Jennings,  H.  S.,  and  Lashley,  K.  S. 

1913.  " Biparental  Inheritance  and  the  Question  of  SexuaUty 
in  Paramecium,"  Jour.  Exp.  ZooL,  XIV,  393-466. 

MiNOT,  C.   S. 

1908.     The  Problem  of  Age,  Growth,  and  Death.     New  York. 

Woodruff,  L.  L. 

1915.  "The  Problem  of  Rejuvenescence  in  Protozoa," 
Biochem.  Bull.,  IV,  371-78. 

191 7.  "Rhythms  and  Endomixis  in  Various  Races  of 
Paramecium  aurelia,''  Biol.  Bull.,    XXXIII,  51-56. 

1917.  "The  Influence  of  General  Environmental  Conditions 
on  the  Periodicity  of  Endomixis  in  Paramecium 
a44relia,"  Biol.  Bull.,  XXXIII,  437-62. 

Woodruff,  L.  L.,  and  Erdmann,  R. 

1914.  "A  Normal  Periodic  Reorganization  Process  without 
Cell-Fusion  in  Paramecium,"  Jour.  Exp.  ZooL,  XVTI, 

425-516. 


CHAPTER  III 
THE  MORPHOLOGY  OF  FERTILIZATION 

Before  beginning  this  subject  it  is  necessary  to  pre- 
pare the  ground  a  Httle  by  certain  considerations  on 
the  germ  cells  themselves.  To  go  at  all  fully  into  their 
characteristics  and  history  would  require  a  volume  in 
itself,  and  the  present  considerations  will  be  strictly 
limited  to  what  is  necessary  for  understanding  fertili- 
zation. 

I.      THE   RELATION   OF  MATURATION   OF  THE   GERM  CELLS 

TO   FERTILIZATION 

After  the  full  growth  of  the  ovum  and  during  or 
immediately  before  fertilization  the  ovum  forms  two 
small  cells,  known  as  the  polar  bodies,  by  a  process  of 
karyokinetic  division  (see  Figs.  4 J,  8c,  gd,  pp.  56,  62, 
and  66).  If  the  first  of  those  cells  divides,  as  sometimes 
happens,  four  cells  are  produced  by  the  fully  grown 
ovum,  three  of  which  are  rudimentary,  viz.,  the  polar 
bodies,  and  the  other  is  the  mature  ovum,  which  is  not 
appreciably  reduced  in  size  by  the' divisions.  The  polar 
bodies  take  no  part  in  development,  but  degenerate, 
though  they  frequently  remain  attached  to  the  egg  for 
a  considerable  period  of  time.  They  are  formed  at  that 
end  of  the  axis,  or  chief  developmental  gradient  of 
the  ovum,  which  is  known  as  the  animal  pole. 

The  polar  bodies  are  essentially  abortive  ova,  and 
the  divisions  by  which  they  are  formed  correspond 
precisely  in  their  nuclear  phenomena  to  the  last  two 

44 


THE  INIORPIIOLOGY  OF  FERTILIZATION  45 

divisions  of  the  spermatocytes  by  which  the  four 
functional  spermatids,  or  immature  spermatozoa,  are 
formed.  These  divisions  in  the  history  of  both  spermat- 
ozoon and  ovum,  known  as  the  maturation  divisions, 
are  concerned  in  reduction  of  the  chromosomes  to  one- 
half  that  characteristic  of  the  species  (haploid  number), 
an  event  that  always  precedes  fertilization;  so  that 
the  union  of  the  two  germ  cells  in  fertilization  restores 
the  species  or  diploid  number. 

The  reduction  divisions  are  preceded  in  both  sper- 
matogenesis and  ovogenesis  by  a  pairing  of  chromo- 
somes in  the  so-called  growth  period  to  form  bivalent 
chromosomes  (tetrads);  it  is  believed  that  the  two 
members  of  a  pair  are  always  maternal  and  paternal, 
respectively,  in  their  origin.  In  one  of  the  reduction 
divisions  the  members  of  each  pair  separate  again  and 
pass  into  different  daughter-cells,  while  in  the  other 
division  each  chromosome  divides  in  the  usual  fashion. 
The  germ  cells  are  thus  prepared,  not  only  by  re- 
duction in  number  of  the  chromosomes,  but  by  differ- 
ential distribution  of  the  latter  for  their  subsequent 
union. 

The  maturation  divisions  of  the  sperm  cells  always 
occur  prior  to  the  special  differentiation  of  this  cell  as 
a  locomotor  cell,  and  thus  long  before  fertilization; 
but  those  of  the  ovum,  which  requires  no  subsequent 
differentiation  to  function  as  a  fully  mature  gamete, 
do  not  occur  until  the  time  of  fertilization  or  immedi- 
ately preceding  it.  Both  kinds  of  cells  lose  their  capa- 
city for  division  after  maturation  unless  they  unite  in 
fertilization;  but  many  ova  lose  this  capacity  prior  to 
maturation,  or   during   the   course   of   the  maturation 


46        *  PROBLEMS  OF  FERTILIZATION 

divisions,  and  either  do  not  begin,  or  do  ^ot  complete, 
their  maturation  divisions,  as  the  case  may  be,  unless 
fertilized.  Thus  the  maturation  and  the  fertilization 
of  the  ovum  frequently  overlap. 

The  following  cases  may  be  recognized:  (i)  The 
ovum  loses  its  capacity  for  division  at  the  end  of  its 
period  of  growth.  The  large  nucleus,  known  as  the 
germinal  vesicle,  undergoes  none  of  the  preparatory 
stages  of  karyokinesis  unless  the  egg  be  fertihzed; 
this  is  the  case,  for  instance,  in  certain  annelids  and 
nematodes,  of  which  the  annelid  Nereis  may  serve  as 
a  type.  (2)  The  ova  of  the  annelid  Chaetopterus,  of 
the  nemertean  Cerebratulus,  and  of  the  lamelhbranch 
Cummingia  pass  through  the  prophases  of  the  first 
maturation  division,  but  the  karyokinetic  process  is 
arrested  in  the  mesophase  of  this  division,  and  the 
ovum  will  die  in  this  condition  unless  fertilized.  (3)  In 
the  case  of  the  ova  of  many  vertebrates  the  first 
polar  body  is  formed,  and  the  prophase  of  the  second 
maturation  division  begins,  but  the  process  then  stops 
unless  the  egg  be  fertilized.  (4)  In  the  echinids  and 
some  other  animals  maturation  is  completed  without 
fertilization. 

In  the  first  three  cases  the  spermatozoon  remains 
more  or  less  quiescent  within  the  egg  during  the  comple- 
tion of  the  maturation  divisions,  and  the  internal  events 
of  fertilization  are  resumed  after  the  formation  of  the 
second  polar  globule.  These  variations  in  the  time  at 
which  the  egg  reaches  the  period  of  inhibition  or  quies- 
cence affect  the  morphological  features  of  fertilization 
in  certain  important  respects;  they  must  be  borne  in 
mind  also  in  the  interpretation  of  experiments. 


THE  MORPHOLOGY  OF  FERTILIZATION  47 

II.      EXTERNAL  AND  •INTERNAL   FERTILIZATION 

The  devices  for  insuring  the  meeting  of  the  sexual 
elements  are  numerous  and  varied.  In  general  the 
conditions  must  be  such  as  to  give  scope  for  movement 
of  the  sex  cells  toward  one  another.  Among  the  Metazoa, 
to  which  our  account  is  limited,  the  ovum  is  incapable 
of  movement  as  a  whole.  The  spermatozoon  is  motile, 
and  its  activities  can  be  maintained  only  in  an  aqueous 
medium  of  suitable  composition  (see  chap,  iv);  sea- 
water  is  such  a  medium  for  most  marine  animals,  and 
the  simplest  conditions  of  union  of  the  germ  cells  are 
found  in  those  marine  animals  that  cast  their  reproductive 
products  into  the  sea-water,  there  to  meet.  Such  are 
most  echinoderms,  many  annelids,  tunicates,  lamel- 
libranchs,  and  bony  fishes.  These  forms  are  the  most 
favorable  for  study  of  fertilization,  for  the  ova,  as  well 
as  the  spermatozoa,  are  produced  in  large  quantity,  and 
the  conditions  and  time  of  their  union  may  be  arbitrarily 
determined.  For  these  reasons  many  of  the  most 
thorough  studies  in  both  the  morphology  and  the  physi- 
ology of  fertilization  have  been  made  on  such  marine 
animals.  But  external  fertilization  is  not  confined  to 
marine  animals;  it  is  also  found  in  fresh- water  fishes 
and  in  anurous  amphibia. 

Many  marine  and  fresh- water  animals  have,  how- 
ever, acquired  methods  of  internal  fertilization,  the 
ovum  being  fertilized  within  the  body  of  the  female; 
and  the  same  is  true  naturally  of  all  terrestrial  animals. 
This  involves  organs  of  copulation,  more  or  less  complex 
secondary  sexual  characters,  and  special  forms  of  mating 
behavior.  In  such  cases  the  meeting  of  the  germ  cells 
is  more  certainly  assured,  and  they  are  produced  in 


48  PROBLEMS  OF  FERTILIZATION 

relatively  small  numbers  as  a' general  rule.  The  study 
of  fertilization  is,  therefore,  usually  more  difficult  techni- 
cally in  such  cases;  and  the  process  is  not  readily  acces- 
sible to  experimental  investigation,  when  it  occurs  in 
the  interior  of  the  body.  For  these  reasons  such  forms 
have  not  been  used  so  extensively  for  study  as  animals 
with  external  fertilization.  Nevertheless  morphological 
studies  of  fertilization,  at  least,  have  been  made  in  nearly 
all  classes  of  the  animal  kingdom. 

III.      THE    SPERMATOZOON 

In  all  animals,  excepting  the  nematodes  and  Crus- 
tacea, the  spermatozoon  is  flagellate;  it  usually  exhibits 
three  readily  distinguishable  parts:  head,  middle  piece, 
and  tail.  Within  this  common  morphological  form 
there  is  the  greatest  possible  diversity  of  organiza- 
tion, so  that  it  is  probable  that  the  spermatozoon 
of  every  species  is  morphologically  distinguishable. 
Such  differences  are  not  usually,  however,  related  in 
any  determinable  way  to  the  processes  of  fertilization 
themselves.  It  is  indeed  probable  that  certain  broad 
features  of  difference  in  organization  are  adaptive  in 
the  sense  that  they  are  related  to  the  conditions  of 
fertilization  in  certain  groups;  but  it  seems  evident 
that  many  of  them  are  results  of  specific  chemical  and 
physical  composition  in  the  given  environment.  It  would 
not  be  profitable,  therefore,  to  examine  their  form  varia- 
tions from  our  point  of  view,  and  the  series  of  figures 
(Fig.  i;  cf.  also  Figs.  3,  4,  6,  7)  may  serve  to  give  an  idea 
of  some  of  the  best-known  variations  in  form  and  size. 

Of  the  three  divisions  of  the  flagellated  spermat- 
ozoon the  head  is  the  most  massive,  containing  the  dense, 


THE  MORPHOLOGY  OF  FERTILIZATION 


49 


concentrated  chromatin  of  the  nucleus,  including  always, 
as  its  history  shows,  the  haploid  number  of  chromosomes. 
It  frequently  bears  anteriorly  a  process  called  for  obvious 


-s 


A 


C 


D 


Fig.  I. — Spermatozoa  of  various  animals:  A,  Diagram  of  the 
flagellate  spermatozoon  (after  E.  B.  Wilson):  i,  perforatorium;  2, 
acrosome;  3,  nucleus;  4,  centrosome  (end  knob);  5,  middle  piece;  6, 
involucre  (envelope)  of  the  tail;  7,  axial  filament;  8,  end  piece.  B, 
Spermatozoon  of  a  crab  (Maia,  after  Grobben).  C,  Spermatozoon  of 
a  bird  {Phyllopneuste,  after  Ballowitz).  D,  Spermatozoon  of  a  sala- 
mander {Amphiuma,  after  McGregor). 

reasons  the  perforatorium.  There  is  usually  no  distin- 
guishable cytoplasmic  mantle  around  the  nucleus, 
though  theoretically  such  a  mantle  should  be  present. 
The.  middle    piece    usually    includes    the    centrosomal 


50  PROBLEMS  OF  FERTILIZATION 

structures  and  mitochondria  of  the  spermatid;  the  tail 
includes  an  axial  filament  which  arises  from  the  cen- 
trosome  in  the  middle  piece  or  at  the  base  of  the 
head;  this  is  surrounded  by  a  protoplasmic  involucre, 
except  for  the  end  piece,  which  is  usually  free. 

IV.   THE  ENTRANCE  OF  THE  SPERMATOZOON  AND  THE 
CORTICAL  CHANGES  OF  THE  EGG 

Under  the  usual  conditions  of  insemination  the 
number  of  spermatozoa  vastly  exceeds  that  of  the  ova. 
Depending  upon  the  concentration  of  the  sperm,  an 
ovum  may  be  associated  with  one  to  several  hundred 
spermatozoa.  As  spermatozoa  tend,  under  uniform 
conditions,  to  form  a  homogeneous  suspension,  i.e., 
to  be  equally  spaced,  their  actual  aggregation  with 
reference  to  the  ova  indicates  some  form  of  mutual 
interaction  which  has  been  variously  interpreted.  An 
old  and  favored  form  of  interpretation  has  been  in 
terms  of  chemotaxis;  the  ovum  was  supposed  to  secrete 
some  substance  that  directs  the  movements  of  the  sper- 
matozoa toward  it.  Many  ova  (e.g.,  echinoderms,  some 
annelids,  etc.)  possess  a  gelatinous  external  layer  which 
seems  to  entangle  spermatozoa  and  hold  them,  and  this 
is  undoubtedly  a  factor  in  such  cases  in  the  aggregation 
of  spermatozoa  around  the  eggs.  There  is  also  the 
possibility  in  some  cases  that  spermatozoa  adhere  to 
ova  and  thus  tend  to  aggregate,  and  this  adhesion  may 
be  specific.     Such  questions  will  be  discussed  later. 

In  most  species  but  a  single  spermatozoon  enters 
the  ovum,  and  as  soon  as  this  happens  others  are  in 
some  way  barred.  Normally  such  ova  are  monospermic, 
but  if  the  ova  are  injured  before  insemination,  two  or 


THE  MORPHOLOGY  OF  FERTILIZATION  51 

more  may  enter  a  considerable  percentage  of  eggs; 
and  the  same  result  may  be  obtained  to  a  lesser  extent 
by  very  heavy  insemination.  Such  polyspermy  in 
normally  monospermic  ova  leads  to  abnormalities  which 
soon  result  in  the  death  of  the  eggs.  There  are,  however, 
certain  species  which  are  normally  polyspermic;  most 
of  these,  as,  e.g.,  Selachia,  reptiles,  and  birds  have  very 
large  ova.     These  questions  will  also  be  discussed  later. 

The  actual  penetration  of  the  spermatozoon  into 
the  ovum  was  first  observed  in  the  sea  urchin  by  Fol, 
in  1876.  (See  discussion  in  chap,  i,  p.  16.)  According 
to  his  account  the  first  spermatozoon  which  comes  in 
contact  with  the  gelatinous  layer  that  surrounds  the 
egg  enters  it  at  once  and  its  point  comes  in  contact 
with  the  egg,  usually  within  a  second  or  two.  The  move- 
ments of  the  tail  then  slacken  and  the  head  of  the  sperm 
elongates  and  enters  the  egg.  The  tail  remains  visible 
for  some  seconds;  then  it  disappears  from  sight.  The 
head  of  the  sperm  forms  a  small  nucleus,  ''male  pro- 
nucleus," within  the  egg.  In  the  starfish  he  described 
a  small  protuberance  of  clear  protoplasm,  the  "fertili- 
zation cone,"  arising  from  the  surface  of  the  egg  at  the 
point  of  contact  of  the  spermatozoon,  lasting  but  a 
few  seconds,  and  appearing  to  aid  in  the  engulfing  of 
the  spermatozoon  in  the  egg. 

In  most  forms  the  process  of  penetration  is  so  rapid 
that  the  details  are  not  readily  observed;  in  Nereis, 
however,  I  have  found  a  form  in  which  the  final  pene- 
tration of  the  spermatozoon  does  not  occur  until  about 
fifty  minutes  after  insemination,  in  which,  therefore, 
all  the  details  of  the  penetration  may  be  observed 
(Figs.   2  and  3).     The  egg  is  provided  with  a  tough 


52 


PROBLEMS  OF  FERTILIZATION 


Fig.  2. — Drawings  from 
photographs  of  Nereis  eggs, 
in  a  suspension  of  India  ink 
in  sea-water  :  a,  before  in- 
semination ;  h,  three  minutes 
after  insemination;  c,  twelve 
minutes  after  insemination, 
a,  The  uninseminated  egg 
is  bounded  by  a  strong  mem- 
brane of  almost  chitinous 
consistency;  within  this  is  a 
cortical  layer  without  yolk 
granules,  and  of  alveolar 
structure ;  h,  immediately 
after  attachment  of  the  sper- 
matozoon (not  shown  in  the 
figure)  the  egg  extrudes  a 
transparent  jelly  from  the 
alveoli  of  the  cortical  layer; 
c,  the  secretion  of  the  jelly 
is  completed;  the  walls  of  the 
emptied  alveoli  of  the  corti- 
cal layer  now  appear  as 
radiating  lines  crossing  the 
peri  vitelline  space.  The  sper- 
matozoon is  seen  (to  right) 
with  a  cone  of  ink  extending 
into  the  jelly  where  its  tail 
lies;  the  protoplasm  of  the 
egg  forms  a  fertilization  cone 
which  crosses  the  perivitelline 
space  and  touches  the  mem- 
brane beneath  the  spermato- 
zoon. 


THE  MORPHOLOGY  OF  FERTILIZATION  53 

vitelline  membrane  beneath  which  is  an  alveolar  cortical 
layer  (Fig.  2a);  the  large  germinal  vesicle  is  central  in 
position;  the  protoplasm  contains  numerous  yolk  spheres 
and  a  broad  equatorial  band  of  refringent  oil  drops. 
When  insemination  takes  place,  a  large  number  of  sper- 
matozoa become  attached  to  the  ovum,  if  the  sperm  is 
present  in  excess.  In  about  two  or  three  minutes  all 
spermatozoa,  with  the  exception  of  one,  which  is  alone 
concerned  in  the  subsequent  fertilization,  begin  to  be 
carried  away  from  the  surface  of  the  egg  by  an  outflow 
of  jelly,  derived  from  the  alveoli  of  the  cortical  layer 
which  are  gradually  emptied,  thus  establishing  a  perivi- 
telline  space  crossed  by  the  delicate  protoplasmic  walls 
of  the  original  alveoli.  A  transparent  fertilization  cone 
then  arises  from  the  inner  wall  of  the  perivitelline  space 
opposite  the  attached  spermatozoon  and  extends  across 
the  space  until  it  touches  the  membrane  at  the  point 
of  attachment  of  the  spermatozoon  (Fig.  2c).  The  per- 
foratorium of  the  spermatozoon  pierces  the  vitelline 
membrane  and  becomes  imbedded  in  the  cone.  These 
phenomena  occupy  about  fifteen  minutes.  The  cone 
then  gradually  flattens  out,  but  stained  sections  show 
that  it  persists  as  a  modified  area  of  the  protoplasm. 
For  about  thirty  minutes  more  no  obvious  changes 
occur.  The  head  of  the  spermatozoon  then  disappears 
rather  abruptly  into  the  ovum,  and  the  tail  and  middle 
piece  are  left  behind  on  the  surface  of  the  vitelline 
membrane. 

Stained  sections  show  the  details  of  the  final  penetra- 
tion of  the  sperm  head  very  beautifully  (Fig.  3).  The 
complex  made  up  of  the  head  of  the  spermatozoon 
and  the  fertilization   cone   act   as   a   unit.     The   cone 


54 


PROBLEMS  OF  FERTILIZATION 


retreats  into  the  interior  of  the  protoplasm  and  the  head 
of  the  spermatozoon  becomes  a  narrow  chromatin 
band  as  it  enters  through  the  minute  aperture  in  the 
viteUine  membrane.  It  is  quite  obvious  that  the  ini- 
tiative in  the  final  act  of  penetration  is  on  the  side  of  the 


I 


i 


a 


c 


i 

i 


Fig.  3. — Penetration  of  the  spermatozoon  in  the  egg  of  Nereis,  from 
sections:  a,  37  minutes  after  insemination;  b,  c,  d,  three  stages  from 
eggs  killed  48^  minutes  after  insemination;  note  that  the  cone  sinks  into 
the  egg  and  draws  the  spermatozoon  after  it;  c,  54  minutes  after  insemi- 
nation; the  head  of  the  spermatozoon  now  entirely  within  the  egg  i? 
contracting;  note  that  the  middle  piece  remains  on  the  membrane;  it 
does  not  enter  the  egg;  the  tail  also  remains  outside. 

ovum.  The  fertilization  cone  is  engulfed  by  the  egg 
protoplasm  and  draws  the  sperm  nucleus  after  it. 
These  events  can  be  understood  by  assuming  that  the 
spermatozoon  causes  a  local  diminution  of  surface 
tension  of  the  egg  in  the  first  place,  thus  causing  an 


THE  MORPHOLOGY  OF  FERTILIZATION  55 

outflow  of  protoplasm  which  is  the  fertihzation  cone; 
that  then  by  coagulation  in  the  cone  the  surface  tension 
of  this  region  rises  until  it  is  overflowed  by  the  surround- 
ing protoplasm  and  sinks  into  the  interior.  The  much 
more  rapid  penetration  in  other  forms  can  be  under- 
stood on  similar  principles. 

In  Nereis  we  have  seen  that  the  head  of  the  sper- 
matozoon alone  enters  the  egg.  The  tail  and  middle 
piece  remain  outside,  l^his  is  exceptional;  in  most 
cases  the  entire  spermatozoon  enters,  as  for  instance 
in  nematodes,  Crustacea,  moUusks,  some  insects,  am- 
phibia, and  mammals  (Fig.  4).  In  some  sea  urchins, 
according  to  descriptions  of  various  authors,  the  tail  is 
left  outside,  but  the  middle  piece  enters  with  the  head. 
The  middle  piece  and  tail  represent  cytoplasmic  elements, 
and  the  head  is  mostly  nuclear  material;  it  would  appear 
from  the  case  of  Nereis  that  the  latter  is  suflicient  of 
itself  for  the  subsequent  events  of  fertilization;  the  middle 
piece  and  tail  are  concerned  primarily  in  accessory  func- 
tions of  fertilization,  such  as  locomotion.  In  the  writer's 
opinion  this  is  their  only  necessary  function,  and  their 
entrance  into  the  egg  in  most  animals  is  incidental; 
however,  this  conclusion  runs  counter  to  certain  con- 
ceptions, and  we  shall  therefore  return  to  its  discussion 
later. 

With  the  attachment  of  the  spermatozoon  to  the 
egg  and  its  penetration  there  are  always  associated 
certain  changes  in  the  cortex  of  the  ovum  which  vary 
considerably  in  their  morphological  expression  in  dif- 
ferent forms.  The  case  of  Nereis  shows  that  actual 
penetration  is  not  necessary  for  these  changes,  but  the 
act  of  penetration  is  so  rapid  in  most  forms  that  it  is 


56 


PROBLEMS  OF  FERTILIZATION 


usually  completed  before  the  cortical  changes  are  evident. 
These  changes  are  the  most  obvious  indicia  of  successful 
insemination,  and,  as  they  are  usually  accepted  in  exper- 
imental parthenogenesis  as  indications  of  initiation  of 


> 


a 


'  *  ii^  ^%. 


X' 


'HH^ 


Fig.  4. — a  and  b,  penetration  of  the  spermatozoon  in  the  oligochaete 
Rhynchelmis  (after  Vejdovsky  and  Mrazek).  Note  the  extensive  yolk- 
free  cone  produced  in  the  egg  cytoplasm,  c,  Spermatozoon  in  the  egg 
of  the  bat  Vespertilio  nodula  (after  Van  der  Stricht).  The  entire  sper- 
matozoon enters,  d,  The  spermatozoon  in  the  egg  of  the  snail  Physa 
fontinalis  (after  Kostanecki  and  Wierzejsky).  The  long  coiled  tail  of 
the  spermatozoon  Ues  in  the  egg  cytoplasm;  sperm  centrosomes  with 
aster  between  tail  and  head. 

development,  it  is  important  to  describe  them.  They 
are  of  great  significance  for  the  physiological  problems 
which  are  considered  later,  and  we  must  examine  them 
with  a  view  to  distinguishing  the  general  from  the  more 
special  features. 


THE  IMORPHOLOGY  OF  FERTILIZATION  57 

It  is  obvious  that  from  a  functional  point  of  view 
all  the  environmental  relations  of  the  ovum  are  involved 
in  the  character  of  the  cortex;  changes  affecting  its 
permeability  must  concern  the  rate  of  cellular  respira- 
tion, access  of  water  and  electrolytes  to  the  interior, 
and  discharge  of  substances  from  the  cell — ^in  short, 
conditions  that  affect  its  metabolism  and  hence  the 
rate  of  developmental  processes.  We  have  seen  in  the 
case  of  Nereis  that  the  cortical  changes  involve  a  dis- 
charge of  material  in  the  form  of  a  clear  jelly  from  the 
cortex  of  the  egg  and  the  consequent  appearance  of  a 
perivitelline  space.  In  most  other  eggs,  if  there  is  a 
discharge  of  material  it  is  of  such  a  nature  as  not  to 
be  morphologically  distinguishable.  It  has  been  inferred 
in  certain  cases  from  the  fact  that  the  diameter  of  the 
egg  appears  to  be  slightly  reduced  following  insemination 
(see  p.  148);  however,  such  measurements  are  pretty 
close  to  the  margin  of  error. 

In  practically  all  eggs  a  perivitelline  space  appears 
between  the  vitelline  membrane  and  the  surface  of  the 
egg  as  a  result  of  insemination.  The  appearances  vary 
here  according  as  there  is,  or  is  not,  a  definite  vitelline 
membrane  prior  to  fertilization.  When  such  a  mem- 
brane is  present,  as  in  all  vertebrates  for  instance,  it 
merely  becomes  more  conspicuous  in  consequence  of 
the  formation  of  the  clear  space.  In  the  frog's  egg  the 
formation  of  the  perivitelline  space,  which  develops 
rapidly  after  insemination,  enables  the  egg  to  rotate 
within  the  membrane  in  accordance  with  the  spccilic 
gravity  of  its  constituent  parts.  In  teleost  eggs  the 
appearance  of  the  space  is  accompanied  by  a  clearing 
of   the   cortical   layer  of  protoplasm,   which   has   been 


58 


PROBLEMS  OF  FERTILIZATION 


interpreted  as  due  to  a  discharge  of  numerous  minute 
refringent  droplets  previously  present.  In  some  other 
cases  a  membrane  appears  to  be  formed  as  a  result 
of  fertilization,  separated  from  the  surface  of  the  egg 
by  a  narrow  perivitelhne  space.  Such  a  membrane  is 
hence  often  called  the  fertilization  membrane.  In  the 
sea  urchin  (Fig.  5)   the  appearance  of  this  membrane 


P     c 


a  h 

Fig.  5. — The  formation  of  the  fertilization  membrane  in  the  egg  of 
the  sea  urchin  Slrongylocentrotiis  purpuratus:  a,  unfertihzed  egg  sur- 
rounded by  spermatozoa;  b,  the  same  egg  about  two  minutes  later  after 
the  entrance  of  the  spermatozoon  (from  Loeb,  Artificial  Parthoiogcncsis 
and  Fertilization,  p.  17;   by  permission  of  the  author). 


is  a  very  obvious  and  rehable  indicator  of  fertilization. 
Fol,  who  first  observed  it,  regarded  it  as  a  device  for 
the  prevention  of  polyspermy,  as  it  begins  to  form  at 
the  point  of  penetration  of  the  spermatozoon  and  spreads 
over  the  entire  periphery  "with  a  rapidity  that  would 
be  inconceivable  if  one  did  not  witness  it."  Whether  it 
is  a  preformed  dehcate  membrane  that  is  merely  elevated 
from  the  surface  of  the  egg  or  a  kind  of  secretion  from 
the  egg  can  hardly  be  answered  from  observation  alone. 


THE  JNIORPHOLOGY  OF  FERTILIZATION  59 

though  the  former  view  appears  to  be  more  probable. 
In  Ascaris  megalocephala  a  very  thick  and  resistant 
fertihzation  membrane  is  formed  as  an  immediate 
result  of  fertilization  (Fig.  6). 

These  changes  have  no  doubt  some  common  physi- 
ological  basis.     Their   special   features  may,   however, 


/ 


/ 


7 


■'*^~  War      ^•^ 


a 


/ 


Fig.  6. — Entrance  of  the  spermatozoon  and  formation  of  the  fertili- 
zation membrane  in  Ascaris  megalocephala:  a,  The  entire  spermatozoon 
within  the  egg;  central  germinal  vesicle  with  tetrads;  the  egg  is  mem- 
braneless.  h,  The  spermatozoon  has  reached  the  center  of  the  egg  and 
its  cytoplasmic  parts  are  disintegrating.  First  maturation  spindle  near 
the  surface.  A  thick  fertilization  membrane,  has  been  formed,  separated 
from  the  egg  by  a  narrow  perivitelline  space. 

also  be  adaptive  in  other  senses.  Thus  the  thick  resist- 
ant fertilization  membrane  of  Ascaris  protects  against 
the  digestive  juices  of  the  host,  the  horse,  and  the  rotation 
of  the  frog's  egg  rendered  possible  by  the  perivitelline 
space,  equalizes  internal  strain  due  to  differences  in 
specific  gravity  of  parts  of  the  egg,  and  is  quite  essential 
to  normal  development. 


6o  PROBLEMS  OF  FERTILIZATION 

V.      THE   INTERNAL  PHENOMENA   OF   FERTILIZATION 

The  morphological  study  of  this  subject  consists  in 
following  the  parts  of  the  spermatozoon  within  the  egg 
and  in  determining  as  far  as  possible  their  relations 
to  constituent  parts  thereof  up  to  the  time  when 
they  can  no  longer  be  separately  distinguished.  The 
nuclear  and  cytoplasmic  parts  of  the  spermatozoon 
have  very  distinct  histories  and  will  therefore,  be  sepa- 
rately treated. 

I.  The  germ  nuclei. — The  nucleus  derived  from  the 
head  of  the  spermatozoon  is  known  as  the  sperm  nucleus 
or  male  pronucleus.  It  is  destined  to  unite  with  the  egg 
nucleus  or  female  pronucleus  derived  from  the  internal 
daughter-chromosome  group  of  the  second  maturation 
division  of  the  egg.  In  those  cases  in  which  the  sper- 
matozoon enters  the  egg  prior  to  or  during  maturation 
the  sperm  nucleus  must  await  the  completion  of  the  matu- 
ration divisions  of  the  egg;  it  has  therefore  more  time  and 
undergoes  more  extensive  changes  before  union  with  the 
egg  nucleus  than  in  those  cases  in  which  maturation  of 
the  egg  is  completed  before  fertilization. 

Immediately  after  penetration  the  head  of  the  Sper- 
matozoon rotates  around  a  transverse  axis  through  i8o° 
(Figs,  ya-f,  So),  so  that  the  base,  which  was  external 
immediately  after  entrance,  becomes  oriented  toward 
the  center  of  the  egg  and  the  apex  is  directed  externally 
(Henking,  Wilson,  Boveri,  Meves,  Lillie,  etc.).  This 
phenomenon  is  very  general,  and  it  may  be  universal. 
No  adequate  explanation  has  been  found  for  it,  and  its 
significance  is  quite  obscure.  It  is,  however,  correlated 
with  the  development  of  the  sperm  aster  which  always 
arises  at  the  base  of  the  sperm  head. 


THE  MORPHOLOGY  OF  FERTILIZATION 


6i 


The  sperm  nucleus  arrives  in  the  egg  with  its  chro- 
matin in  the  most  condensed  condition.  The  nucleus 
then  begins  to  enlarge  by  imbibition  of  fluid  and  tends 


\ 


a 


§ 

b 


(^V*    £■ 


Fig,  7. — Fertilization  of  the  egg  of  the  sea  urchin  Toxopncustcs 
(after  E.  B.  Wilson):  a,  the  spermatozoon;  h,  c,  the  sperm  head  and 
middle  piece  immediately  after  entrance;  tail  apparently  absent; 
beginning  of  rotation  c;  d,  rotation  half-way  completed;  origin  of  sperm 
aster;  e,  rotation  completed,  middle  piece  separated  from  sperm  centro- 
some;  /,  g,  approach  of  the  germ  nuclei;  growth  of  the  sperm  aster;  //, 
meeting  of  the  germ  nuclei;  division  of  the  sperm  aster;  i,  first  seg- 
mentation nucleus  in  which  the  sperm  and  egg  components  are  readily 
distinguished. 


'"'^\^'KS^W^"'"^'7'^  .'■ 


internal 


a  h 

Fig.  8. — Sections     of     successive     stages     showing     the 
phenomena  of  fertihzation  in  Nereis. 

a,  Section  of  an  egg  54  minutes  after  insemination.  Cf.  Fig.  3^. 
The  head  of  the  spermatozoon  has  rotated,  the  sperm  nucleus  is  becom- 
ing rounded,  and  the  sperm  aster  is  beginning  to  arise  opposite  to  the 
cone.  The  latter  marks  the  apex  of  the  sperm  head.  The  first  polar 
body  is  fully  formed,  h,  The  sperm  cone-nucleus-aster  complex  of  an 
egg  64  minutes  after  insemination.  The  sperm  cone  is  now  separated 
from  the  nucleus,  and  is  destined  soon  to  disappear.  The  sperm  aster 
and  centrosome  better  developed. 


Fig.  8  (continued). — c,  67  minutes  after  insemination;  stage  of 
anaphase  of  second  maturation  division.  The  sperm  aster  has  divided, 
forming  an  amphiaster.  d,  jy  minutes  after  insemination.  The  sperm 
nucleus  Hes  to  the  left  below;  the  sperm  amphiaster  has  become  reduced. 
The  egg  nucleus,  which  is  formed  by  fusion  of  chromosome  vesicles, 
is  represented  by  two  still  unfused  parts  to  the  right  above. 


THE  MORPHOLOGY  OF  FERTILIZATION 


63 


to  become  vesicular.  I'his  change,  however,  proceeds 
relatively  slowly  during  the  maturation  of  the  egg; 
in  some  cases,  in  which  the  enlargement  of  the  sperm 
nucleus  begins  in  the  early  stages  of  maturation  of  the 
egg,  there  is  a  halt  or  actual  decrease  in  size  during  the 
later  stages  until  the  second  polar  body  is  formed, 
when  the  definitive  increase  in  size  ensues  (e.g.,  U^iio, 
according  to  Lillie,  1895). 
In  such  cases  the  sperm 
nucleus  after  penetrating 
a  short  distance  may  cease 
to  move  {Undo),  or  it  may 
penetrate  to  the  center  of 
the  egg  and  then  come 
to  rest  (Nereis,  Fig.  8^). 
After  maturation  is  com- 
pleted the  sperm  nucleus 
and  the  egg  nucleus  en- 
large in  practically  equal 
tempo  and  come  together 
in  a  predetemiined  region 


.7  •»       "IkN  ■ 


lv«^: 


^:^p 

y<j^fs 

■  '•; 

1 

Fig.  8  {continued). — e,  Somewhat 
later.  The  two  germ  nuclei  have 
fused  and  the  first  cleavage  spindle 
is  forming  with  unequal  centers. 
The  chromosomes  of  sperm  origin 
lie  below;  those  of  egg  origin,  above. 
Only  a  few  of  the  latter  fell  in  the 
plane  of  section. 


of  the  egg  (Figs.  M,  e,  9). 
They  are  then  usually  of  the  same  size  and  appearance, 
so  that  they  can  be  distinguished  only  by  their  positions 
or  associations. 

When  maturation  of  the  egg  is  completed  before 
fertilization,  as  in  the  sea  urchin,  the  egg  nucleus  and 
the  sperm  nucleus  proceed  directly  to  the  place  of 
meeting,  and  the  sperm  nucleus  is  much  smaller  than  the 
egg  nucleus  at  the  time  of  union  (Fig.  7  g,  //,  i).  The 
chromatin  is,  however,  much  more  condensed  and  is 
equal  in  quantity  to  that  of  the  egg  nucleus;  if  the  nuclei 


64  PROBLEMS  OF  FERTILIZATION 

are  prevented  from  meeting  rapidly,  e.g.,  by  the  use  of 
anaesthetics,  as  in  E.  B.  Wilson's  experiments  (1901), 
the  sperm  nucleus  may  grow  to  the  size  of  the  egg  nucleus, 
and,  after  recovery  from  the  anaesthetic  effect,  the  two 
equal  nuclei  unite. 

We  have,  therefore,  to  consider  two  questions  to 
the  extent  that  morphological  observation  admits: 
(a)  What  determines  the  movements  of  the  germ  nuclei 
within  the  egg  and  their  union  ?  (b)  What  is  the  nature 
of  the  union  quantitatively  and  qualitatively  ? 

a)  As  regards  the  first  question,  Roux,  in  1883-87, 
resolved  the  movements  of  the  sperm  nucleus  w^ithin 
the  egg  into  two  components,  which  he  called  the  pene- 
tration path  and  the  copulation  path.  His  observations 
were  made  on  the  frog's  egg,  in  which  the  spermatozoon 
leaves  behind  it  a  trail  of  pigment,  marking  out  its 
path,  which  is  usually  curved  or  exhibits  an  angle.  He 
conceived  the  first  part  of  the  path  to  be  a  continua- 
tion of  the  direction  of  penetration;  the  second  part 
of  the  path  he  conceived  to  be  determined  by  an  attrac- 
tion between  the  egg  nucleus  and  the  sperm  nucleus. 
That  there  is  an  energy  of  penetration  of  the  sper- 
matozoon which  persists  in  the  same  direction  after 
entrance  into  the  egg  is  scarcely  tenable,  because 
the  penetration  itself  is  not  a  result  of  the  locomotor 
energy  of  the  spermatozoon;  there  is  also  no  reason 
to  assume  that  the  nuclei  as  such  exert  attraction  on 
one  another.  Such  an  assumption  has  no  basis  in 
fact  beyond  the  actual  meeting  of  the  germ  nuclei, 
which  can  equally  well  be  explained  on  other  more 
reasonable  and  less  mystical  grounds.  The  view  has 
been  presented  that  the  movements  of  the  sperm  nucleus 


THE  MORPHOLOGY  OF  FERTILIZATION  65 

are  brought  about  by  the  sperm  aster,   the  fibers  of 
which  were  supposed  to  act  as  contractile  elements. 

The  movements  of  the  germ  nuclei  within  the  egg* 
depend  on  conditions  of  equilibrium  of  the  various  cell 
constituents  which  constitute  a  definitely  ordered  stream 
of  events.  The  localization  jo(  the  nucleus  within  the 
cell  is  always  determinate.  We  have  therefore  to  con- 
ceive that,  as  both  sperm  nucleus  and  egg  nucleus  are  in 
physiological  relations  to  the  same  mass  of  cytoplasm, 
which  is  preparing  to  divide,  they  must  reach  the  same 
position  of  equihbrium  within  the  cell,  and  hence  of 
necessity  meet.  Their  coming  together  is  due,  not 
to  mutual  attraction,  but  to  independent  movements 
toward  the  same  part  of  the  developing  egg.  This  tend- 
ency cannot,  however,  manifest  itself  until  after  matura- 
tion is  completed;  hence  the  movements  of  the  sperm 
nucleus  prior  to  the  completion  of  maturation- are  not 
always  directed  toward  the  ultimate  place  of  union  of 
the  germ  nuclei,  being  under  the  influence  of  a  different 
condition  of  equilibrium  of  the  egg  cytoplasm.  The 
curved  or  bent  path  of  the  spermatozoon  in  certain  cases 
follows  from  this,  and  it  is  not  found  in  the  echinids, 
where  maturation  is  complete  before  fertilization. 

h)  The  two  nuclei  thus  united  may  fuse  together 
to  form  a  single  nucleus  called  the  first  segmentation 
nucleus,  in  which  it  may  be  difficult  to  distinguish  the 
two  components  for  a  certain  period  of  time.  But  in 
many,  perhaps  most,  cases  the  changes  preparatory  to  the 
first  cleavage  of  the  egg  begin  before  such  a  fusion  occurs, 
and  in  these  cases  it  is  easy  to  determine  that  each 
germ  nucleus  contributes  the  same  number  of  chromo- 
somes to  the  first  segmentation  spindle  (Fig.  ga,  b,  d). 


66 


PROBLEMS  OF  FERTILIZATION 


These  are  the  only  components  of  the  germ  nuclei  that 
can  be  traced  morphologically  beyond  this  time.  Even 
in  those  cases  in  which  a  typical  first  segmentation 
nucleus  occurs  (Fig.  p)  it  is  equally  certain  that  the 
maternal  and  paternal  chromatins  form  equal  chromo- 


a 


&t> 


%^ 


Fig.  g. — (After  Boveri):  a,  Pronuclei  of  Ascaris  megalocephala, 
approaching  between  the  attraction  spheres  of  the  first  cleavage  spindle, 
each  containing  two  chromosomes,  b,  The  first  cleavage  spindle  fully 
formed;  it  contains  four  chromosomes  which  are  shown  in  a  polar  view  of 
the  same  spindle  in  the  small  figure  to  the  right  above.  Two  of  these 
chromosomes  are  of  maternal  and  two  of  paternal  origin,  c,  Meeting 
of  germ  nuclei  of  Pterotrachea  (pteropod) ;  each  contains  sixteen  chromo- 
somes, d,  Formation  of  the  first  cleavage  spindle  in  Pterotrachea.  The 
maternal  and  paternal  chromosome  groups  are  separate. 


THE  IMORPHOLOGY  OF  FERTILIZATION  67 

some  groups  upon  the  first  segmentation  spindle, 
because  they  are  usually  sHghtly  separated  and  it  is 
known  that  each  germ  nucleus  contains  only  the  hap- 
loid  number  of  chromosomes,  whereas  the  first  segmen- 
tation spindle  has  always  the  diploid  number. 

Van  Beneden  (1883)  was  the  first  to  discover  this 
invariable  law  of  fertiHzation;  he  discovered  that  in 
Ascaris  megaloce phala  each  germ  nucleus  produces  two 
chromosomes,  so  that  the  first  segmentation  spindle 
contains  four  (Fig.  9).  These  divide  longitudinally  in 
the  usual  way  so  that  the  first  two  cells  each  receive 
four  daughter-chromosomes,  two  of  maternal  origin 
and  two  of  paternal  origin;  he  assumed  that  this  condi- 
tion was  transmitted  to  all  subsequent  generations  of 
cells,  which  thus  possess  nuclei  of  biparental  origin. 
This  involves  the  idea  that  maternal  and  paternal  chro- 
matins remain  distinct  within  each  cell  throughout  the 
life-history,  and  thus  a  basis  is  furnished  for  explaining 
both  the  intimate  intermingling  of  paternal  character- 
istics in  the  offspring  and  the  independent  behavior 
of  such  characteristics  in  heredity.  Subsequent  studies 
of  the  behavior  of  the  germ  nuclei  include  a  large  num- 
ber of  forms  and  have  demonstrated  the  same  principles 
to  be  universal. 

We  must  dwell  further  upon  this  question.  Un- 
doubtedly the  most  fundamental  fact  which  the  mor- 
phological study  of  fertilization  has  revealed  is  the 
equivalence  of  the  germ  nuclei  with  reference  to  the 
chromosomes.  This  is  a  definite  and  undeniable  posi- 
tive result  which  is  in  perfect  agreement  with  the  equiva- 
lence of  the  sexes  in  inheritance.  In  all  other  respects 
the  germ  cells  are  differentiated  in  opposite  directions; 


68  PROBLEMS  OF  FERTILIZATION 

in  this  one  particular  they  are  demonstrably  alike. 
No  wonder  that  this  determination  has  furnished  the 
foundation  for  the  most  elaborate  chromosome  theories 
from  the  time  of  Weismann  until  the  present  day,  which 
have  been  supported,  changed,  and  rectified  by  the 
most  painstaking  investigations  of  chromosome  be- 
havior in  all  stages  of  the  cycle  of  the  germ  cells. 

This  morphological  and  genetic  equivalence  is  also 
physiological  in  the  sense  that  either  germ  nucleus  is 
adequate  in  itself  for  purposes  of  development.  This 
is  proved  for  the  egg  nucleus  by  artificial  partheno- 
genesis, and  for  the  sperm  nucleus  by  those  experiments 
in  which  an  enucleated  fragment  of  an  egg  fertilized  by 
a  single  spermatozoon  has  been  proved  to  develop 
{merogony,  see  p.  162). 

Differences  between  the  germ  nuclei  on  the  morpho- 
logical side  have  been  shown  to  occur  between  those 
chromosomes  which  are  concerned  in  sex  determination 
(Morrill,  1910;  Mulsow,  1912;  see  Fig.  10);  on  the  genetic 
side  differences  exist,  undeterminable  morphologically, 
which  depend  on  the  genetic  history  of  the  individual 
and  which  are  of  an  entirely  similar  order  in  both  germ 
nuclei.  The  foundation  of  all  genetic  theory  of  sexually 
produced  organisms  thus  rests  upon  the  demonstrated 
equivalence  of  the  germ  nuclei. 

2.  Other  constituents  of  the  spermatozoon  in  the  egg. — 
In  addition  to  the  nucleus  the  spermatozoon  usually 
introduces  certain  cytoplasmic  constituents  into  the 
egg,  but  as  contrasted  with  the  nucleus  there  has  been 
the  greatest  difficulty  in  tracing  the  fate  and  determin- 
ing the  significance  in  fertilization  of  these  elements, 
which,  moreover,  vary  greatly  in   different   groups   of 


THE  MORPHOLOGY  OF  FERTILIZATION 


69 


animals.  In  some  cases,  as  in  Ascaris,  the  quantity 
of  cytoplasm  thus  introduced  is  very  considerable 
(Fig.  6a);  in  other  cases,  as  in  Nereis  (Fig.  3),  none  can 


\ 


a  b 

Fig.  10. — Fertilization  of  a  nematode  {Ancyracanthus  cyslidicola) 
(after  Mulsow) :  In  each  figure  the  upper  nucleus  is  the  egg  nucleus,  the 
lower  the  sperm  nucleus.  In  both  figures  the  egg  nucleus  contains  six 
chromosomes;  in  a  the  sperm  nucleus  contains  five  chromosomes,  in 
h  six.  The  combination  6+5  in  a  gives  the  male  number,  eleven;  the 
combination  6  +  6  in  h  gives  the  female  number,  twelve.  The  two 
classes  of  spermatozoa  are  hence  regarded  as  male  producing  and  female 
producing  respectively. 

be  demonstrated  to  occur.  It  is  generally  believed 
with  good  reason  that  the  perforatorium  and  tail  have 
no  further  significance  in  fertilization  after  penetration 
is  once  achieved;    in  any  event  they  are  not  traceable 


70  PROBLEMS  OF  FERTILIZATION 

after  a  very  early  stage.  Attention  has  been  focused, 
largely  on  theoretical  grounds,  on  two  constituents  in- 
timately associated  with  the  middle  piece  of  the  sper- 
matozoon, viz.,  the  centrosome  and  the  mitochondria 
derived  from  the  spermatid. 

a)  The  sperm  centrosome:  Shortly  after  penetra- 
tion of  the  spermatozoon  in  very  many  animals  an 
aster  arises  in  association  with  the  sperm  nucleus 
(Figs,  ^d,  7,  8);  it  is  centered  at  the  base  of  the  sperm 
head  in  all  cases  in  which  its  actual  beginning  has  been 
traced,  and  it  has  therefore  been  supposed  to  be  caused 
by  the  middle  piece  of  the  spermatozoon;  a  central 
body  soon  appears  in  the  aster;  this  is  the  sperm  centro- 
some, which  has  been  regarded,  therefore,  as  derived 
from  the  middle  piece;  these  observations  have  been 
correlated  with  the  histogenesis  of  the  spermatozoon,  in 
which  it  has  been  shown  in  many  cases  that  the  centro- 
some (or  part  of  the  centrosome)  of  the  spermatid  is 
located  in  the  middle  piece.  It  was  therefore  concluded 
that  the  centrosome  of  the  sperm  aster  within  the  egg 
is  derived  from  the  centrosome  of  the  spermatid.  It 
was  further  determined  in  a  considerable  number  of 
cases  that  the  sperm  aster  by  division  forms  an  am- 
phiaster  which  produces  the  first  cleavage  of  the 
egg  (Figs.  7,  8).  From  reasoning  of  this  kind  Boveri 
deduced  his  famous  theory  of  fertihzation  that  the  initia- 
tion of  development  is  due  essentially  to  the  introduc- 
tion of  an  active  division  center  into  an  egg  devoid  of 
centrosomes,  and  hence  without  capacity  for  division. 
This  conception  obviously  involves  a  whole  theory  of 
cell  division,  and  reciprocally  such  a  theory  should  be 
supported  or  weakened  by  the  facts  of  fertilization. 


THE  MORPHOLOGY  OF  FERTILIZATION  71 

The  exact  facts  about  the  origin  of  the  sperm  aster 
in  fertilization  should  therefore  be  most  carefully  ascer- 
tained. In  the  sea  urchins,  where  the  process  was  first 
carefully  studied,  the  aster  begins  to  be  visible  shortly 
after  the  rotation  of  the  sperm  head  has  begun.  It  is 
focused  at  the  base  of  the  sperm  head,  thus  in  the 
region  of  the  middle  piece;  the  latter  is  not,  however, 
in  the  center,  but  to  one  side,  of  the  aster,  as  Meves's 
very  detailed  study  shows  (cf .  Fig.  ye) .  A  differentiated 
centrosome  is  not  demonstrable  in  the  center  of  the 
forming  aster  (Fig.  7^),  or  at  most,  as  Boveri  says,  it 
is  such  an  immeasurably  small  granule  that  it  can  be 
seen  only  in  especially  favorable  cases,  and  then  only 
because  of  its  position;  it  is  never  to  be  seen  prior  to 
the  origin  of  the  aster.  In  Nereis,  in  which  the  middle 
piece  of  the  sperm  does  not  enter  the  egg,  the  sperm 
aster  appears  at  first  to  be  focused  at  the  base  of  the 
sperm  nucleus  itself  and  only  by  degrees  separates 
from  it  and  acquires  a  distinct  centrosome  (Figs.  8a,  Z>,  c). 
I  have,  moreover,  been  able,  by  application  of  a  strong 
centrifugal  force  to  the  inseminated  eggs,  to  remove  not 
only  the  middle  piece  but  also  variable  parts  of  the  base 
of  the  sperm  head  itself  before  penetration,  so  that  a 
reduced  sperm  nucleus  forms  after  penetration.  In  the 
case  of  such  nuclei  asters  form  at  the  base,  opposite  to 
the  perforatorium,  in  the  usual  way,  which  are  more  or 
less  proportional  in  size  to  the  nuclear  fragment  concerned 
(Fig.  11).  This  shows  that  the  sperm  nucleus  itself  in  this 
case  has  the  capacity  to  induce  localized  aster  formation 
in  the  egg  cytoplasm;  this  reaction  might  be  conceived 
to  be  due  to  a  specific  centrosome  substance  contained 
within  the  nucleus,  but  for  this  there  is  no  evidence. 


72 


PROBLEMS  OF  FERTILIZATION 


The  formation  of  asters  is  postponed  in  some  animals 
until  about  the  time  of  meeting  of  the  germ  nuclei,  and 
then  it  is  often  difficult  to  determine  whether  or  not 


iT.' 


I 


a 


m 


m 


i 


• 


Fig.  II. — Effects  of  centrifugal  force  on  penetration  of  the  sper- 
matozoon in  Nereis:  a,  b,  and  c  show  removal  of  varying  portions  of  the 
sperm  head  before  penetration;  d  shows  the  later  history  of  an  egg  into 
which  a  minute  portion  of  the  spermatozoon  has  entered;  this  part  has 
produced  a  sperm  aster  and  centrosome,  although  it  represents  only 
a  fraction  of  the  apical  end  of  the  sperm  head  similar  to  the  piece 
shown  in  c. 

the  sperm  components  are  alone  concerned  in  it.  In 
Crepidula  (Conklin)  and  Unio  (Lillie)  there  is  evidence 
that  each  germ  nucleus  causes  formation  of  one  aster 
of  the  first  cleavage.  • 


THE  MORPHOLOGY  OF  FERTILIZATION  73 

In  those  cases  in  which  the  history  of  the  sperm 
aster  is  clearly  shown  throughout  (echinids,  many 
annelids  and  mollusks,  ascidians,  etc.),  the  central 
body  of  the  aster  soon  divides  in  two  centrosomes  which 
move  apart  with  consequent  formation  of  an  amphi- 
aster  (Figs.  4,  7,  8),  which  becomes  the  achromatic 
part  of  the  first  cleavage  spindle.  In  echinids  (Fig.  7), 
in  which  maturation  is  completed  and  the  egg  nucleus 
formed  prior  to  insemination,  the  division  takes  place 
about  the  time  of  meeting  of  the  germ  nuclei,  for  there 
are  no  preparatory  changes  remaining  to  be  accom- 
plished by  the  egg  and  the  two  nuclei  therefore  unite 
very  rapidly.  The  plane  of  the  separation  in  this  case 
is  at  right  angles  to  the  line  uniting  the  centers  of  the 
two  germ  nuclei.  In  those  cases  in  which  the  egg 
has  part  of  the  maturation  process  still  to  complete 
the  sperm  amphiaster  remains  more  or  less  quiescent 
during  maturation  (Fig.  8),  but  it  may  entirely  dis- 
appear (Unio)  or  diminish  to  a  variable  extent  (e.g.. 
Nereis),  and  the  asters  of  the  first  cleavage  spindle 
are  to  this  extent  new  formations,  though  certainly  in 
some  cases,  and  possibly  in  others,  formed  around  the 
original  centers. 

The  morphological  variations  are  very  numerous 
with  respect  to  the  genetic  behavior  of  the  first  cleavage 
amphiaster  when  we  compare  all  the  various  species 
studied.  As  the  physiological  foundation  must  be  sup- 
posed to  be  uniform  we  can  interpret  the  morphological 
variations  as  due  to  time  differences  in  the  components 
of  the  physiological  reactions  in  different  species. 

When  we  find  that  the  sperm  nucleus  behaves  dif- 
ferently in   respect   to   aster   formation   from    the   egg 


74  PROBLEMS  OF  FERTILIZATION 

nucleus  within  the  same  body  of  cytoplasm  we  must 
attribute  this  either  to  the  presence  of  some  specific 
substance  originally  present  with  the  former,  but  not 
with  the  latter,  or  to  some  other  differential  organi- 
zation of  the  substances  of  the  nuclei  concerned.  Boveri 
took  the  former  alternative  and  formulated  it  in  terms 
of  a  morphological  theory  of  cell  division.  He  thus 
brought  under  one  point  of  view  the  origin  and  locaH- 
zation  of  the  sperm  aster  and  the  phenomena  of  cell 
division,  so  that  the  theory  of  fertilization  became  part 
of  a  theory  of  cell  division.  He  stressed  cell  division 
as  the  fundamental  factor  of  development  and  over- 
looked the  more  immediate  and  essential  result  of 
activation  of  metabolism. 

The  centrosome  theory  of  cell  division  has  not, 
however,  maintained  itself  in  the  face  of  advancing 
knowledge  of  cell  morphology  and  physiology.  Boveri' s 
theory  of  fertilization  would  thus  lose  much  of  its  sig- 
nificance even  if  it  were  demonstrated  that  the  sperm 
centrosome  is  a  derivative  of  the  spermatid  centrosome, 
and  this  is  far  from  being  the  case.  Not  only  is  there 
a  hiatus  in  the  history,  but  the  experiments  on  Nereis 
previously  cited  show  that  the  sperm  nucleus  itself 
contains  material  for  inciting  formation  of  a  sperm 
aster  and  its  centrosome.  It  is  possible  to  attribute 
this  to  specific  centrosome  material  contained  within 
the  nucleus,  if  the  centrosome  theory  of  cell  division 
is  adhered  to,  but  it  is  also  possible  to  explain  it  in  a 
more  physiological  manner  to  be  considered  hereafter 
(p.  265).  A  finer  morphological  analysis  is  no  doubt 
possible  and  will  be  useful,  but  we  must  recognize  the 
fact  that  in  fertilization  we  are  dealing  with  a  physio- 


THE  MORPHOLOGY  OF  FERTILIZATION  75 

logical  process  that  requires  experimental  methods  for 
its  solution. 

h)  The  mitochondria  in  fertilization:  The  other 
cytoplasmic  element  that  has  been  claimed  to  play 
an  important  role  in  fertihzation  is  the  mitochondria 
(called  plastochondria  by  Meves),  which  form  such  an 
apparently  important  constituent  of  all  classes  of  cells. 
The  special  protagonist  of  the  significance  of  this  sub- 
stance in  fertilization  is  Meves,  who  maintains  that 
it  is  concerned  in  the  transmission  of  hereditary  char- 
acteristics, basing  this  view  on  the  part  that  he  believes 
it  to  play  in  protoplasmic  differentiation.  He  found 
an  apparently  very  demonstrative  case  in  A  scan's 
megalocephala  (Meves,  191 1),  the  spermatozoa  of  which 
contain  large  numbers  of  mitochondrial  granules  in 
the  large  cytoplasmic  body  surrounding  the  nucleus 
(cf.  Fig.  6).  The  entire  spermatozoon  penetrates  in 
this  case,  and  the  mitochondrial  granules  continue  to 
surround  the  nucleus  long  after  penetration ;  by  degrees 
they  become  intermingled  with  the  mitochondria  of  the 
egg,  and  Meves  even  hazarded  the  conjecture  that  they 
possibly  united  by  pairing  with  the  mitochondrial  gran- 
ules of  the  egg;  he  therefore  considered  his  view  that  the 
mitochondria  play  an  important  role  in  heredity  justified. 

Pursuing  the  matter  farther  Meves  (1914)  made  an 
exceedingly  careful  study  of  the  fate  of  the  middle 
piece  of  the  spermatozoon  in  the  fertilized  eggs  of 
echinids.  By  a  veritable  triumph  of  technique  he  found 
that  this  minute  fragment  could  be  traced  intact  into 
one  of  the  first  two  cells;  in  successive  cell  divisions  it 
always  passes  intact  into  one  of  the  daughter-cells 
only,  and  may  be  found  in  the  eight-celled  stage  either 


76  PROBLEMS  OF  FERTILIZATION 

in  one  of  the  animal  or  one  of  the  vegetative  quartet; 
he  even  traced  it  to  the  thirty-two-celled  stage.  Thus 
it  is  not  broken  up  and  distributed  to  all  of  the  cells, 
as  the  theory  that  it  represents  a  substratum  for  bearing 
heredity  factors  would  require,  nor  does  it  exhibit  any 
signs  of  activity.  In  the  egg  of  an  ascidian  (Phallusia) 
the  same  author  (1913)  could  follow  the  sperm  plasto- 
chondria  through  part  of  the  fertilization  stages,  but 
then  lost  sight  of  them.  Van  der  Stricht,  in  the  bat, 
and  Lams,  in  the  guinea-pig,  found  that  the  tail  of  the 
spermatozoon  and  connecting  piece  which  carries  plasto- 
chondria  pass  into  one  only  of  the  first  two  cells. 
Finally,  in  Nereis,  according  to  my  own  observations, 
the  middle  piece  of  the  spermatozoon,  which  is  usually 
supposed  to  carry  the  plastochondria,  does  not  enter 
the  egg  at  all. 

Whatever  may  be  the  function  of  the  mitochondria 
in  cell  physiology  it  must  be  admitted  that  the  study 
of  fertilization  has  shown  no  reason  for  the  assumption 
that  their  introduction  into  the  egg  by  the  sperm  is 
necessary  for  the  transmission  of  paternal  character- 
istics. The  variable  quantity  in  different  cases  and  the 
distribution  to  single  blastomeres  in  certain  cases 
exclude  the  hypothesis  that  they  have  any  specific 
paternal  hereditary  effect.  There  is  no  reason  to  deny 
that  sperm  mitochondria  function  in  the  egg  when 
present,  but  if  so  it  is  probable  that  they  are  not  dif- 
ferentiated in  their  chemical  composition  or  genetic 
behavior  from  the  mitochondria  of  the  egg  itself. 

3.  The  egg  cytoplasm. — The  egg  cytoplasm  and  its 
inclusions  constitute  an  exclusively  maternal  material 
which  determines  many  of  the  characters  of  early  embry- 


THE  MORPHOLOGY  OF  FERTILIZATION  77 

onic  stages;  such  characters  are  therefore  exclusively 
maternal.  The  materials  of  the  cytoplasm  are,  how- 
ever, being  constantly  consumed  in  the  metabolism, 
and  the  process  of  renewal  and  increase  of  such  materials 
involves  interaction  of  nucleus  and  cytoplasm;  there- 
fore the  purely  maternal  cytoplasm  soon  disappears 
and  is  replaced  by  cytoplasm  formed  under  the  influence 
of  the  biparental  zygote  nucleus.  Maternal  cytoplasmic 
characters  cannot  therefore  survive  long  in  the  life- 
history,  unless  the  cytoplasm  contains  elements  either 
that  survive  as  such  or  that  increase  independently  of 
the  nucleus.  Mitochondria  granules  may  be  such  ele- 
ments, and  in  plants  also  plastids,  including  the  chloro- 
phyll grains  of  chloroplasts.  There  may  be  conceivably 
other  chemical  substances  that  have  a  purely  cyto- 
plasmic history,  but  for  this  we  have  little  evidence. 
We  have,  however,  adequate  cytological  grounds  in 
such  persistent  elements  of  composition  of  the  egg 
cytoplasm  for  the  explanation  of  the  rather  rare  cases 
of  exclusively  maternal  inheritance  from  a  zygote 
known  to  the  geneticists.  Purely  paternal  inheritance 
probably  does  not  exist  in  any  regularly  formed  zygote, 
and  this  constitutes  an  independent  line  of  negative 
evidence  against  cytoplasmic  inheritance  from  the  male 
side. 

VI.      POLYSPERMY 

The  ova  so  far  considered  are  normally  monospermic; 
there  are,  however,  certain  ova  into  which  more  than 
one  spermatozoon  enters  normally,  and  practically  all 
ova  may  become  polyspermic  under  abnormal  condi- 
tions. We  may  thus  distinguish  normal,  or  physio- 
logical, and  pathological  polyspermy. 


78  PROBLEMS  OF  FERTILIZATION 

I.  Physiological  polyspermy. — Physiological  poly- 
spermy occurs  in  vertebrates  possessing  eggs  of  large 
size  devoid  of  a  strong  membrane  and  micropyle,  in 
which  penetration  of  the  spermatozoon  may  occur  at 
any  spot  within  a  large  area.  We  may  conceive  in 
such  cases  that  the  protective  mechanism  against 
penetration  of  supernumerary  spermatozoa,  which  be- 
gins to  form  at  the  point  of  penetration  and  spreads, 
does  not  extend  itself  with  sufficient  rapidity  to  protect 
the  entire  fertilizable  surface  from  other  spermatozoa. 
The  large  eggs  of  sharks,  of  some  amphibia,  of  reptiles, 
and  of  birds  are  thus  polyspermic.  Polyspermy  occurs 
in  the  eggs  of  several  classes  of  insects  which  possess 
several  micropyles  (Henking,  1891).  Among  animals 
possessing  small  eggs  it  occurs  apparently  only  in  Bry- 
ozoa,  in  which  the  spermatozoa  are  united  in  bundles 
(Bonnevie,  1907). 

In  all  cases  of  normal  polyspermy  only  one  of  the 
sperm  nuclei  formed  from  the  entering  sperm  heads 
unites  with  the  egg  nucleus,  and  the  supernumerary 
sperm  nuclei  are  disposed  of  in  certain  ways.  Thus 
the  fertilization  in  such  cases  is  finally  monospermic. 
In  the  fertilization  of  the  pigeon,  for  instance,  from 
about  twelve  to  twenty-five  spermatozoa  enter  the  ger- 
minal disk  as  soon  as  the  ovum  is  released  from  the  ovary 
(Harper,  1904).  The  second  maturation  division  occurs 
after  this,  and  during  this  time  the  sperm  heads  accu- 
mulate in  a  ring  of  protoplasm  surrounding  the  matura- 
tion spindle  at  some  distance  from  it  (Fig.  12).  After 
completion  of  this  division  and  formation  of  the  egg 
nucleus  one  of  the  sperm  nuclei  moves  centrally  and 
unites  in '  the  usual  way  with  the  egg  nucleus,  while 


THE  MORPHOLOGY  OF  FERTILIZATION  79 

the  supernumerary  sperm  nuclei  move  away  from  the 
center  as  though  repelled  and  accumulate  in  the  periph- 
eral periblast.  Here  they  undergo  division  and  pro- 
duce cell  areas  in  the  periblast,  which  are  a  conspicuous 

sp  /v: 

"  ^.*.  •■so*-'*        "  ^  -  'v^"^      "  "J^**"^'  ''.  ■'•■•-  ^  J  //     C*       r 

:. ' /  i  -  :  • .  /  V  .;  ;■    J^o  '  IS^'""^  t; ' 


.     -.-„  ,,    .  ..  .,-. .  -         ,-'        "•• 


■     O   - 
Co  " 


V   \- 


■  »-•  »  : 


A      _,«#    «   '  ^ 


y/!.V 


>  f^* 


/•\-  ,o 


Fig,  12. — Part  of  a  horizontal  section  of  the  germinal  disk  of  a 
pigeon's  egg  freed  from  the  ovary,  but  not  yet  in  the  oviduct.  First 
maturation  spindle  {M.S.  i)  cut  transversely  in  the  center;  sperm  nuclei 
{Sp.N.)  surrounding  it  (after  E.  H.  Harper). 

feature  of  the  development  up  to  about  the  thirty- 
two-celled  stage,  at  which  time,  according  to  Miss 
Blount,  they  begin  to  degenerate,  and  soon  entirely 
disappear.  The  segmentation  nucleus  in  this  case  is 
thus  formed  of  the  union  of  the  egg  nucleus  and  a  single 
sperm  nucleus  in  the  usual  way,  and  all  nuclei  of  the 
embryo  are  derived  from  this  by  karyokinetic  division. 
In  Selachia  the  phenomena  are  similar,  but  Riickert 
maintains  that  the  supernumerary  sperm  nuclei  persist 


8o  PROBLEMS  OF  FERTILIZATION 

in  the  periblast  which  forms  extra-embryonic  tissue. 
It  may  be,  however,  that  this  conclusion  is  due  to  con- 
fusion with  other  nuclei  of  the  periblast  derived  from 
the  segmentation  nucleus. 

The  peripheral  migration  of  the  supernumerary 
nuclei  furnishes  an  interesting  problem.  Miss  Blount 
has  suggested  in  her  study  of  these  nuclei  in  the  pigeon 
that  the  phenomenon  can  be  understood  on  the  simple 
assumption  that  migration  of  the  sperm  nuclei  is  al- 
ways into  unfertilized  protoplasm,  i.e.,  such  as  has  not 
yet  been  modified  by  the  sperm  nuclei;  the  central 
protoplasm  surrounding  the  egg  nucleus  is  thus  modi- 
fied by  the  first  sperm  nucleus  that  moves  into  it,  but 
the  peripheral  protoplasm  is  still  virgin  soil. 

There  would  thus  seem  to  be  no  advantage  connected 
with  physiological  polyspermy;  at  the  most  it  is  harm- 
less, and  merely  represents  a  condition  in  which  the 
final  determination  of  the  successful  spermatozoon  is 
completed  within  the  egg.  Such  eggs  have  in  some 
way  overcome  the  usually  harmful  effects  of  polyspermy. 
Bonnevie  (1907),  however,  is  of  the  opinion  that  in 
Bryozoa  at  least  it  is  significant  for  the  maintenance 
of  the  organism;  she  suggests  that  the  supernumerary 
spermatozoa  furnish  extra-nuclear  chromatin  of  physio- 
logical importance.  There  is,  however,  no  adequate 
foundation  for  such  a  view  at  present. 

2.  Pathological  polyspermy. — Eggs  normally  mono- 
spermic  may  be  entered  by  more  than  one  spermatozoon 
if  they  are  allowed  to  become  stale  before  insemination; 
the  same  result  may  be  attained  by  exposing  them  to  the 
action  of  various  injurious  substances,  such  as  chloroform, 
chloral  hydrate,  cocaine,  nicotine,  strychnine,   quinine. 


THE  MORPHOLOGY  OF  FERTILIZATION  8i 

and  many  others,  in  appropriate  concentrations  for 
proper  periods  of  time.  Moreover,  a  very  heavy  insemi- 
nation of  any  normal  lot  of  eggs  will  usually  yield  a  small 
percentage  of  polyspermy.  The  number  of  spermatozoa 
that  may  enter  under  such  circumstances  may  vary  from 
two  to  a  considerable  number.  The  first  student  of  this 
subject,  Fol,  in  1877,  determined  for  the  starfish  and 
sea  urchin  that  polyspermic  eggs  divide  in  more  than 
two  cells  at  the  first  cleavage  and  their  subsequent 
development  is  never  normal. 

In  1887  O.  and  R.  Hertwig  pubhshed  a  detailed 
study  of  polyspermy  in  the  sea  urchin;  each  sperm 
nucleus  forms  an  aster  which  subsequently  divides  to 
form  an  amphiaster.  If  only  two  sperm  nuclei  are 
present  both  unite  with  the  egg  nucleus,  and  the  two 
amphiasters  produce  a  four-poled  karyokinetic  figure, 
or  tetraster;  the  egg  divides  simultaneously  into  four 
cells,  but  the  subsequent  division  of  the  cells  is  always 
in  two  each.  Triasters  sometimes  form,  owing  to  fusion 
of  two  asters,  and  a  simultaneous  division  of  the  egg 
into  three  cells  follows.  If  more  spermatozoa  enter,  all 
sperm  nuclei  do  not  necessarily  unite  with  the  egg 
nucleus;  two  or  more  may  unite  with  the  egg  nucleus 
and  a  multipolar  figure  results;  the  other  sperm  amphi- 
asters then  associate  themselves  with  this  figure  and 
very  complex  karyokinetic  systems  result. 

In  the  frog  (Herlant,  191 1),  each  sperm  nucleus  forms 
an  aster,  as  in  the  sea  urchin,  but  the  egg  nucleus  unites 
with  only  one  of  the  sperm  nuclei;  the  supernumerary 
sperm  nuclei  form  karyokinetic  figures  also.  Thus 
in  the  case  of  dispermy  two  karyokinetic  figures  result, 
one  of  which  contains  the  chromosomes  of  the  egg  and 


82  PROBLEMS  OF  FERTILIZATION 

one  sperm  nucleus,  the  other  only  the  chromosomes  of 
the  supernumerary  sperm  nucleus.  In  the  case  of  tri- 
spermy  we  have  three  karyokinetic  figures — one  diploid, 
two  haploid.  The  dispermic  egg  divides  in  two  cells 
and  the  trispermic  in  three,  but  each  of  these  cells 
is  binucleated.  In  the  dispermic  egg  one  nucleus  of 
each  cell  is  diploid,  the  other  haploid;  in  trispermic 
eggs  this  apphes  to  two  of  the  cells,  but  the  two 
nuclei  of  the  third  cell  are  both  haploid.  In  subse- 
quent divisions  the  proportions  of  diploid  nuclei  is  main- 
tained. 

In  the  sea  urchin,  in  the  frog,  and  also  in  all  other 
cases  so  far  as  known,  pathological  polyspermic  eggs 
produce  abnormal  embryos,  which  soon  die.  Boverl 
has  made  a  most  careful  and  interesting  analysis  of 
conditions  in  the  sea  urchin,  which  led  him  to  the 
conclusion  that  the  ill  effects  are  due  in  this  case  to 
abnormal  distribution  of  the  chromosomes.  Taking 
the  simplest  case  of  dispermy  he  shows  that  the  distri- 
bution of  chromosomes  in  the  tetraster  is  highly  irreg- 
ular and  a  matter  of  chance,  from  which  it  results 
that  the  four  nuclei  formed  have  diff'erent  numbers  of 
chromosomes.  This  would  not  in  itself  account  for 
the  abnormal  results,  because  it  is  known  that  half 
the  diploid  chromosome  number  is  sufficient  for  normal 
development,  and  he  could  show  that  the  number  in 
each  nucleus  exceeds  this  number  on  the  average. 
From  this  he  argues  that  the  chromosome  composition 
of  the  nuclei  must  be  on  the  average  inadequate;  that 
not  merely  a  given  number  of  chromosomes,  but  a  defi- 
nite qualitative  composition  of  the  chromosome  group, 
is  necessary  for   normal   development.     He   thus   con- 


THE  MORPHOLOGY  OF  FERTILIZATION  83 

ceives  that  the  chromosomes  of  each  germ  nucleus  are 
quahtatively  differentiated,  and  that  a  full  representa- 
tion of  chromosome  qualities  is  necessary  for  normal 
development.  His  experiments  constitute  an  argument 
for  quahtative  differences  of  chromosomes  which  has 
been  generally  accepted.  The  result  is  reached  by  ex- 
exclusion  of  other  possible  causes  of  abnormality. 
This  subject  leads  into  certain  phases  of  cytology  that 
do  not  belong  in  our  field. 

The  case  of  the  frog  is  somewhat  different,  in  that 
the  nuclei  are  either  definitely  diploid  or  haploid. 
Herlant  comes  to  the  conclusion  that  the  cause  of  death 
in  this  case  is  the  different  size  of  the  nuclei  and  their 
associated  cell  bodies  in  the  same  embryo,  which  renders 
normal  functioning  impossible,  and  other  more  obscure 
probable  causes  of  disharmony  associated  with  this 
principle. 

For  the  subject  of  morphology  of  fertilization  the 
study  of  polyspermy  is  significant  in  two  principal 
respects:  (i)  It  furnishes  the  demonstration  that  the 
sperm  nucleus  is  different  from  the  egg  nucleus,  owing 
'  either  to  association  of  a  centrosome  with  it  or  for  other 
cause;  because  we  find  that  each  sperm  nucleus  pro- 
duces a  definite  effect  on  the  cytoplasm  of  the  egg,  the 
formation  of  an  aster,  which  the  egg  nucleus  itself 
does  not  produce  in  the  cases  studied.  (2)  The  in- 
evitable pathological  result,  when  more  than  one  sperm 
nucleus  is  concerned  in  the  development,  furnishes 
important  evidence  for  the  nuclear  theory  of  heredity. 
On  the  physiological  side  the  study  of  polyspermy  is 
significant  from  other  aspects,  which  we  shall  examine 
later. 


84  PROBLEMS  OF  FERTILIZATION 

VII.      FERTILIZATION   AND    SYMMETRY 

In  1854  the  English  naturahst  Newport,  who  had 
devoted  much  time  to  a  series  of  briUiant  observations 
and  experiments  on  the  fertihzation  of  the  ova  of 
Amphibia,  reported  "that  the  first  cleft  of  the  yolk 
is  in  a  line  with  the  point  of  the  egg  artificially  impreg- 
nated, and  that  the  head  of  the  young  frog  is  turned  to- 
ward the  same  point."  He  thus  made  the  discovery, 
which  has  since  been  confirmed  by  Roux,  Schulze,  Bra- 
chet,  and  others,  that  there  is  a  definite  relation  between 
the  point  of  penetration  of  the  spermatozoon  and  the  sym- 
metry of  the  resulting  embryo  in  the  case  of  the  frog.  As 
a  result  of  these  studies  it  has  been  shown  that  the  pene- 
tration of  the  spermatozoon  determines  that  meridianal 
plane  of  the  polarized  egg  in  which  the  first  plane  of 
cleavage  usually  forms,  and  in  which  the  axis  of  the 
embryo  probably  always  arises.  Subsequent  research 
has  also  confirmed  Newport's  statement  that  the  head 
of  the  embryo  is  turned  toward  the  point  of  penetration. 

Roux  attempted  the  first  theoretical  explanation 
of  this  fact  on  the  basis  that  the  path  of  the  spermat- 
ozoon determines  the  plane  of  apposition  of  the  germ 
nuclei.  The  division  of  the  zygote  nucleus  must,  he 
thought,  run  at  right  angles  to  the  plane  of  apposition 
in  order  to  secure  impartial  distribution  of  maternal 
and  paternal  nuclear  constituents  to  the  two  cells. 
Thus  the  first  plane  of  cleavage  would  pass  along  the 
penetration  path  of  the  spermatozoon  and  approxi- 
mately through  the  point  of  entrance.  In  certain  cases, 
however,  it  was  shown  that  the  first  plane  of  cleavage 
did  not  coincide  either  with  the  plane  of  symmetry  or 
with  the  fertilization  meridian.     Brachet's  experiments 


THE  MORPHOLOGY  OF  FERTILIZATION  85 

on  the  frog's  egg  have  shown  that  in  such  cases  the  plane 
of  symmetry  and  the  fertihzation  meridian  neverthe- 
less coincide.  Roux's  theory  therefore  does  not  hold 
except  for  the  direction  of  the  first  plane  of  cleavage, 
which  in  the  frog  may  not  coincide  with  the  plane  of 
symmetry. 

There  is,  however,  no  necessary  relation  between 
the  fertilization  meridian  and  the  plane  of  symmetry, 
because  polyspermic  eggs  develop  a  plane  of  symmetry, 
and  so  also  do  parthenogenetic  eggs.  The  relationship 
which  has  been  shown  to  exist  in  certain  cases  must 
therefore  depend  upon  a  certain  time  relationship  in 
the  course  of  the  two  processes.  The  influences  radi- 
ating from  the  spermatozoon  establish  a  gradient  from 
its  original  eccentric  position,  which  may  influence  the 
direction  of  the  plane  of  symmetry  in  which  there  is 
also  a  gradient,  if  its  determination  is  synchronous,  as 
in  the  frog. 

In  the  case  of  the  annelid  Nereis  the  observations 
of  Just  (191 2)  show  that  the  plane  of  symmetry  is  at 
right  angles  to  the  fertilization  meridian.  Here  again 
we  have  obviously  an  interaction  of  two  distinct  gra- 
dient processes,  which,  however,  attain  a  different  equi- 
librium from  the  frog.  The  data  on  this  subject  are 
few,  owing  to  the  difficulty  of  making  the  necessary 
determinations.  The  ova  of  many  animals  are  definitely 
bilaterally  symmetrical  before  fertihzation,  which  has 
therefore  nothing  to  do  with  its  determination.  The  con- 
clusion would  therefore  seem  to  be  that,  when  the 
determination  of  symmetry  and  fertihzation  overlap, 
the  former  may  be  affected  as  to  its  orientation  by  the 
latter. 


86  PROBLEMS  OF  FERTILIZATION 

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1909.  "The  Early  Development  of  the  Pigeon's  Egg,  with 
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CONKLIN,  E.   G. 

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FoL,  Hermann. 

1877.     See  references  at  end  of  chapter  i. 

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Griffin,  Bradney  B. 

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Harper,  E.  H. 

1904.  "The  Fertilization  and  Early  Development  of  the 
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Henking,  H. 

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Hertwig,  O.  and  R. 

1887.     See  references  at  end  of  chapter  i. 

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Just,  E.  E. 

191 2.  "The  Relation  of  the  First  Cleavage  Plane  to  the 
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King,  Helen  Dean. 

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lentiginosus,"  Jour,  of  Morph.,  XVII,  293-342. 

Kostanecki,  K.,  and  Wierzejsky,  a. 

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Jour,  of  Morph.,  XVII,  227-92. 

191 2.  "Studies  of  Fertilization  in  Nereis:  III,  The  Mor- 
phology of  the  Normal  Fertilization  of  Nereis;  IV, 
The  Fertilizing  Power  of  Portions  of  the  Spermato- 
zoon," Jour.  Exp.  Zool.,  XII,  413-76. 

Mead,  A.  D. 

1898.  "The  Origin  and  Behavior  of  the  Centrosomes  in 
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Meves,  F. 

191 1.  "Ueber  die  Beteiligung  der  Plastochondrien  an  der 
Befruchtung  des  Eies  von  Ascaris  megalocephala,'^ 
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76,  pp.  683-713. 

191 2.  "Verfolgung  des  sogenannten  Mittelstiickes  des 
Echinidenspermiums  im  befruchteten  Ei  bis  zum 
Ende  der  ersten  Furchungsteilung,"  ibid..  Band  80, 
pp.  81-123. 

1913.  "Ueber  das  Verhalten  des  Plastomatischen  Bestand- 
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von  Phallusia  mammillata,''  ibid.,  Band  82,  Abt.  II, 
pp.  215-60. 


THE  MORPHOLOGY  OF  FERTILIZATION  89 

Meves,  F. 

1914.     "Verfolgimg   des   Mittclsttickes  des   Echinidcnsper- 
miums  durch  die  ersten  Zcllgenerationcn  des  bcfruch- 
teten  Lies,"  ibid.,  Band  85,  Abt.  II,  pp   1-8 
Morrill,  Charles  V. 

1910.     "The  Chromosomes  in  the  Oogenesis,  Fertilization  and 
Cleavage  of  Corcid  Hemiptcrar   Biol.   Bull.,   XIX 
79-126.  ' 

Mulsow,  Karl. 

1912.     "Die  Chromosomencyklus  bei  Ancyracanlhus  cystidl- 
colar  Arch.jiir  Zcllf.,  Band  9,  pp.  67,-n. 
Newport,  George. 

1854.     See  references  at  end  of  chapter  i. 
Oppel,  Albert. 

1892.     Die  Befruchtung  des  Reptilieneies,"  Arch.  Jilr  mikr 

Roux,  W    ^^"''^'  "'"^  Entwickelungsgesch.,  Band  39,  PP-  215-90. 

1887.     "Die    Bestimmung    der    Medianebene    des    Frosch- 

embryos  durch  die  Copulationsrichtung  des  Eikernes 

und  des  Spermakernes,"  Arch.  f.  mikr.  Anal.,  Band 

29,  pp.  157-212. 

RUCKERT,   J. 

1895.  "Ueber  das  Selbstandigbleiben  der  vaterlichen  und 
miitterlichen  Kernsubstanz  wahrend  der  ersten  Ent- 
wickelung  des  befruchteten  Cyclops  Eies,"  Arch, 
mikr.  Anal.,  Band  45,  pp.  339-69. 

1899.  -Die  erste  Entwickelung  des  Eies  der  Elasmobran- 
chier,"  Festschrift  zum  70.  Geburtstag  von  C.  v. 
Kupffer  (pp.  581-704)  (see  earher  references  here).' 
Jena. 

SCHULZE,   O. 

1899.  "Ueber  das  erste  Auftreten  der  bikUcralen  Sym- 
metric im  Verlauf  der  Entwickelung,"  Arch,  fiir 
mikr.  Anal,  und  Entwickelungsgesch.,  Band  55,  pp. 
202-31. 

SOBOTTA,   J. 

1895.  "Die  Befruchtung  und  Furchung  des  Eies  der  Maus," 
Arch.  mikr.  Anal.,  Band  45,  pp.  15-92. 


90  PROBLEMS  OF  FERTILIZATION 

Van  Beneden,  E. 

1883.     See  references  at  end  of  chapter  i. 
Van  der  Stricht,  O. 

1898.  ''La  formation  des  deux  globules  polaires  et  I'appari- 
tion  des  spermocentres  dans  I'oeuf  de  Thysanozooii 
brocchi''  Arch,  de  bioL,  T.  15,  pp.  367-461. 

1902.  ''Le  spermatozoide  dans  I'oeuf  de  chauve-souris 
{V.  noctula),'"  Verh.  d.  Anal.  GeselL,  pp.  163-68. 

Vejdovsky,  F.,  and  Mrazek,  a. 

1903.  "Umbildung  des  Cytoplasma  wahrend  der  Befruch- 
tung  und  Zelltheilung.  Nach  Untersuchungen  am 
Rhynchelmis-Eie,"  Arch.  Jiir  mikr.  Anat.  und  Enl- 
wickeliingsgesch.,  Band  62,  pp.  431-579. 

Wheeler,  W.  M. 

1897.  "The  Maturation,  Fecundation  and  Early  Cleavage 
of  Myzostoma  glabrum,  Leuckart,"  Arch,  de  biol., 
T.  15,  pp.  1-77. 

Wilson,  E.  B. 

1895.  "Archoplasma,  Centrosome  and  Chromatin  in  the 
Sea  Urchin  Egg,"  Jour,  of  Morph.,  XI,  443-78. 

1896.  The  Cell  in  Development  and  Inheritance.  New  York: 
Macmillan. 

1901.     "Experimental  Studies  in  Cytology:  II,  Some   Phe- 
nomena of  Fertilization  and  Cell-Division  in  Etherized 
Eggs,"  Arch,  fiir  Entwickelungsmech.,  Band  13,  pp. 
353-95- 
Wilson,  E.  B.,  and  Leaming,  E. 

1895.  An  Atlas  of  Fertilization  and  Karyokinesis  of  the 
Ovum.    New  York. 

Wilson  E.  B.,  and  Mathews,  A.  P. 

1895.  "Maturation,  Fertilization  and  Polarity  in  the 
Echinoderm  Egg,"  etc.,  Jour,  of  Morph.,  X,  319-42. 


CHAPTER  IV 

THE  PHYSIOLOGY  OF  THE  SPERMATOZOON 

I.      INTRODUCTION 

What  are  the  forms  of  behavior  of  flagellated  sper- 
matozoa ?  What  conditions  are  optimum  ?  What 
changes  in  environment  are  significant  ?  Such  ques- 
tions are  vital  for  the  study  of  the  physiology  of  fertili- 
zation; but  the  subject  has  not  been  studied  with  the 
attention  that  its  importance  deserves.  Such  knowl- 
edge as  we  possess  demonstrates  that  spermatozoa  are 
exceedingly  sensitive  organisms  in  certain  respects. 
With  reference  to  the  reaction  (i.e.,  H  ion  equilibrium) 
of  sea-water,  for  instance,  the  spermatozoa  of  certain 
marine  forms  are  as  delicate  indicators  as  we  possess. 
The  general  belief  appears  to  be  that  in  the  medium 
in  which  fertilization  takes  place  spermatozoa  are  in  a 
condition  of  ceaseless  random  activity;  but  are  their 
movements  entirely  undirected  ?  Do  they  indeed  dilYer 
from  other  organisms  in  exhibiting  no  directiveness 
of  behavior  ?     To  what  stimuli  do  they  respond  ? 

II.      GENERAL 

Spermatozoa  are  almost  invariably  immobile  in  the 
testis  and  the  efferent  ducts.  They  become  active  in 
the  medium  in  which  insemination  takes  place.  Among 
marine  animals  there  are  great  variations  in  this  re- 
spect. Thus  the  spermatozoa  of  most  annelids  and 
sea  urchins  and  many  other  forms  become  exceedingly 

91 


92  PROBLEMS  OF  FERTILIZATION 

active  in  sea-water;  but  in  the  case  of  the  starfish 
they  usually  do  not  activate  greatly  in  sea-water, 
save  in  the  presence  of  excess  of  OH  ions  or  secretions 
of  the  eggs  of  the  same  species,  a  matter  to  which  we 
shall  return.  In  mammals  the  spermatozoa  are  active 
in  the  ejaculate  which  contains  secretion  of  the  pros- 
tate and  other  glands.  Sperm  taken  from  the  epidid- 
ymis will  activate  in  physiological  salt  solution. 

Spermatozoa  are  probably  incapable  of  receiving 
nourishment  outside  of  the  gonad  after  they  are  fully 
differentiated;  certainly  in  the  case  of  all  forms  with 
external  insemination  there  is  no  opportunity  for  the 
restitution  of  substance.  We  must  therefore  regard 
these  cells  as  charged  with  their  full  available  store  of 
energy  in  the  testis  and  their  capacity  for  locomotion 
as  thus  determined  and  limited.  They  therefore  have 
a  strictly  limited  period  of  life,  the  duration  of  which 
will  be  determined  by  their  activity.  The  store  of 
energy  is  saved  when  they  are  motionless  and  expended 
when  in  motion.  Thus  we  find  that  sperm  suspensions 
will  retain  their  fertihzing  power  for  a  relatively  long 
time  if  activity  is  reduced,  and  will  lose  it  relatively 
rapidly  if  activity  is  great.  Cohn  (1918)  has  deter- 
mined that  the  total  CO2  production  of  spermatozoa 
is  the  same  whether  their  life  be  long  or  short. 

The  amount  of  energy  produced  has  not  been  meas- 
ured, but  it  is  necessary  to  describe  it  as  surprisingly 
large;  a  certain  degree  of  motion  may  be  kept  up  for 
hours  at  a  time.  The  source  of  the  energy  is  certainly 
in  the  cytoplasm,  as  the  highly  condensed  nucleus  is 
in  a  condition  which  all  cytological  experience  inter- 
prets as  a  state  of  quiescence.     If  in  the  cytoplasm, 


PHYSIOLOGY  OF  THE  SPERMATOZOON  93 

the  cytological  data  would  suggest  the  mitochondria 
as  a  source  of  energy.  This  is,  however,  purely  specu- 
lative, for  the  subject  has  not  been  studied  from  this 
point  of  view;  but  the  universal  presence  of  mitochon- 
dria in  the  sperm  suggests  some  important  function,  and 
they  are  almost  certainly  not  heredity  material. 

The  locomotion  of  flagellated  spermatozoa  seems  to 
be  essentially  similar  in  all  animals.  The  spermat- 
ozoa of  Nereis  in  their  free  movements  through  the 
water  describe  spiral  paths  with  rather  close-set  turns. 
As  soon  as  a  spermatozoon  comes  in  contact  with  a 
surface  it  tends  to  move  round  and  round  in  circles 
in  contact  with  the  surface  in  anticlockwise  direction; 
the  forward  component  of  the  locomotion  is  largely 
eliminated  under  these  conditions.  The  movement  is 
due  to  successive  beats  of  the  tail,  and  it  is  an  interest- 
ing fact  that  under  certain  conditions  of  aggregation 
the  successive  beats  of  the  aggregated  spermatozoa 
may  become  synchronous;  the  rate  is  about  120  a 
minute   at    20°  C.    under   such    circumstances    (LiUie, 

1913)- 

The  spermatozoa  of  sea  urchins  also  swim  spirally 

when  freely  suspended.     "The  spirals  may  be  so  steep 

that    the   spermatozoa    appear    to    swim    in    ahiiost   a 

straight  line,   and  they   then  move  relatively  rapidly 

across  the  field  of  the  microscope.     On  the  other  hand, 

the  incline  of   the   spiral  may  be  so  gentle  that  the 

spermatozoa  appear  to  be  swimming  almost  in  circles" 

(Buller,  1902).     The  spermatozoa  of  sand  dollars  behave 

similarly.     Ballowitz   (1890)   and   Dewitz  (1885,   1886) 

describe  similar  forms  of  behavior  for  various  insects, 

Massart  (1888,  1889)  ^or  Amphibia. 


94  PROBLEMS  OF  FERTILIZATION 

The  conditions  under  which  spermatozoa  are  usu- 
ally observed  are  not  favorable  for  observation  of 
unimpeded  locomotion;  the  material  is  usually  observed 
in  the  form  of  suspensions  of  greater  or  less  density  in 
which  the  individual  spermatozoa  are  continually  colhd- 
ing  with  one  another  and  with  the  walls  of  the  chamber 
in  which  they  are  confined.  Under  such  circumstances 
the  distribution  of  active  spermatozoa  should  be  uni- 
form, like  the  molecules  of  a  gas,  and  this  condition  is 
found  in  perfectly  fresh  suspensions.  However,  it 
usually  does  not  last  long  and  various  forms  of  aggre- 
gation result. 

The  spermatozoa  of  Nereis  suspended  in  sea-water 
give  a  very  striking  reaction  which  illustrates  the  point. 
If  a  drop  of  dry  sperm  from  a  mature  Nereis  is  mixed 
in  about  6  c.c.  of  sea- water  in  a  Syracuse  watch  crys- 
tal it  makes  a  uniformly  milky  suspension;  in  a  few 
seconds  clouds  begin  to  appear,  and  in  fifteen  to  forty- 
five  seconds  these  usually  draw  together  in  white  solid- 
looking  masses  uniformly  spaced  through  the  fluid 
(Fig.  13).  The  intervening  fluid  becomes  quite  clear 
and  the  masses  quickly  settle  on  the  bottom.  The 
rate  of  formation  of  these  masses  and  their  number  and 
size  depend  on  condition  of  the  animal  furnishing  the 
sperm,  temperature,  "freshness"  of  the  sperm,  reaction 
of  the  medium,  etc.  Sperm  suspensions  of  most  animals 
do  not,  however,  exhibit  such  marked  aggregations. 

III.      BEHAVIOR   OF   SPERMATOZOA 

The  function  of  the  spermatozoon  is  apparently 
bound  up  with  its  capacity  for  locomotion;  it  is  prob- 
able   that    immobilized    sperm    will    not    fertilize,    in 


PHYSIOLOGY  OF  THE  SPERMATOZOON  95 

spite  of  Schiicking's  (1903)  statement  to  the  contrary. 
Loeb  (19 1 5)  has  shown  that  spermatozoa  will  not 
fertilize  in  the  presence  of  an  immobilizing  concentra- 
tion of  NaCN,  no  matter  how  concentrated  the  sperm, 
*' while  the  same  sperm  when  it  revives  from  the  effect 


Fig.  13. — Photograph  of  the  aggregation  of  a  sperm  suspension  of 
Nereis,  taken  90  seconds  after  mixing  the  suspension  (natural  size). 

of  NaCN  fertilizes  the  same  eggs  at  once."  The 
experiment  is  not  conclusive  proof  of  the  necessity  of 
motility  for  fertilization,  because  the  block  in  the  ferti- 
lization reaction  might  conceivably  be  due  to  some  other 
effect  of  the  NaCN.  The  experiment,  however,  ren- 
ders it  very  probable  that  motility  of  the  sperm  is 
necessary. 


96  PROBLEMS  OF  FERTILIZATION 

But  motility  is  not  the  exclusive  requirement; 
the  spermatozoon  must  exhibit  other  definite  forms  of 
behavior  with  reference  to  the  egg.  It  has,  however, 
proved  impossible  to  analyze  the  behavior  of  the  sper- 
matozoon by  direct  observation  of  fertilization.  Hence 
it  is  desirable  to  observe  the  various  forms  of  behavior 
of  spermatozoa  apart  from  the  egg  in  the  expectation 
of  being  able  to  utilize  the  information  thus  gained  in 
the  study  of  fertilization. 

Spermatozoa  are  highly  specialized  cells  with  ref- 
erence to  behavior  as  well  as  to  structure  and  function. 
Their  principal  behavior  reactions  appear  to  be  with 
reference  to  temperature,  contact,  and  chemical  stimuli. 
They  are,  indeed,  like  all  cells,  also  sensitive  to  changes  in 
osmotic  pressure;  but  we  have  no  evidence  of  reaction  to 
light  or  gravitation.  Their  behavior  consists  in  changes 
in  rate  of  activity,  in  maintaining  contact  with  surfaces 
either  at  rest  or  not,  in  alterations  in  direction  of  loco- 
motion, and  in  adhesion  to  one  another  or  other  surfaces. 

I.  Light,  Gravity,  Osmosis,  Temperature 

By  way  of  clearing  the  ground  we  may  first  consider 
the  least  known,  or  least  effective,  possible  sources  of 
stimulation. 

a)  Light. — So  far  as  known,  animal  spermatozoa  are 
quite  indifferent  to  light  conditions.  The  chlorophyll- 
bearing  spermatozoa  of  many  plants,  however,  exhibit 
definite  responses  to  illumination.  These  are  entirely 
lacking  in  animals,  but  we  cannot  suppose  that  the 
metabolism  remains  the  same  under  all  light  conditions, 
and  no  doubt  careful  investigation  would  show  some 
effect  of  presence  or  absence  of  light  or  of  rays  of  differ- 
ent length  on  the  activity  of  spermatozoa. 


PHYSIOLOGY  OF  THE  SPERMATOZOON  97 

b)  Gravitation. — Similarly  animal  spermatozoa  arc 
not  known  to  exhibit  any  definite  reaction  to  gravi- 
tation. The  spermatozoa  of  marine  forms  are  of  some- 
what greater  specific  gravity  than  the  sea- water,  hence 
they  tend  to  sink  when  at  rest,  or  may  be  precipitated 
by  the  centrifuge. 

c)  Osmotic  pressure. — Spermatozoa  of  Nereis  are- 
apparently  more  sensitive  to  increase  than  to  decrease 
of  osmotic  pressure.  They  are  fairly  active  in  5  c.c. 
of  sea-water  plus  2.5  c.c.  of  distilled  water,  but  are 
paralyzed  in  5  c.c.  of  sea-water  plus  i  c.c.  of  2|M.  NaCl 
(Lillie,  1913). 

d)  Temperature. — Temperature  affects  the  rate  of 
movement  of  spermatozoa;  it  is  rather  difficult  to  meas- 
ure rate  of  movement  directly,  but  in  the  case  of  Nereis 
the  aggregation  reaction  described  on  page  94,  which 
is  a  function  of  the  activity,  gives  us  a  means  of  ready 
observation : 

At  13°  C.     No  aggregations  form. 

At  1 5°  C.     Slight  signs  of  aggregation  in  four  minutes. 

At  i8°-i9°  C.  Aggregation  in  from  two  to  four 
minutes;  much  fewer  in  number  than  at  higher  temper- 
atures. 

At  20.5°  C.     Numerous  aggregations  in  one  minute. 

At  23.5°  C.  Y^et  more  numerous  aggregations  in 
thirty  seconds. 

At  26.5°  C.  No  aggregations  form  until  the  tem- 
perature falls  to  about  23°. 

Thus  in  this  case  temperatures  from  20  to  23.5°  C. 
are  optimum.  At  15°  the  movements  of  the  sper- 
matozoa are  too  slow  to  produce  the  aggregation  re- 
action,   and    at    26.5°,    although    the   movements   are 


gS  PROBLEMS  OF  FERTILIZATION 

extremely  active,  they  are  apparently  unco-ordinated, 
so  that  the  aggregation  reaction  is  not  given. 

As  a  general  principle  the  rate  of  activity  of  sper- 
matozoa is  a  function  of  the  temperature,  like  other 
biological  processes,  and  the  temperature  range  varies 
with  the  species. 

2 .  Contact 

All  kinds  of  spermatozoa  studied  exhibit  contact 
reactions.  Dewitz  (1885,  1886)  appears  to  have  been 
the  first  to  study  this  subject;  he  showed  that  the 
spermatozoa  of  the  cockroach  maintain  contact  with 
sohd  bodies  and  free  surfaces  of  liquids;  he  studied 
their  thigmotactic  rotations  on  the  surface  of  the  egg, 
which  finally  result  in  the  penetration  of  the  micropyle 
of  this  hard-shelled  ^gg.  In  a  drop  beneath  a  raised 
cover  slip  they  divide  in  two  groups,  one  in  contact 
with  the  floor,  the  other  with  the  roof  of  the  inclosed 
space,  and  are  absent  in  between.  They  are  in  constant 
motion,  describing  circles  of  varying  diameters  always  in 
the  same  sense  (anticlockwise),  with  reference  to  the 
surface  of  contact;  but  these  two  groups  appear  to 
revolve  in  opposite  directions  under  the  microscope, 
owing  to  the  direction  of  observation. 

Massart  (1888,  1889)  studied  the  reactions  of  the 
spermatozoa  of  the  frog  and  showed  that  in  a  drop  be- 
neath a  raised  cover  slip  they  also  divide  in  two  actively 
rotating  groups,  one  in  contact  with  the  shde  below  and 
the  other  with  the  cover  slip  above;  similarly  in  a 
hanging  drop  they  accumulate  in  contact  with  the 
glass  above  and  the  free  surface  of  the  drop  below. 
Buller  (1902)  states  that  when  sea  urchin  spermatozoa 
*'come  in  contact  with  a  surface  they  either  become 


PHYSIOLOGY  OF  THE  SPERMATOZOON  99 

fixed  to  it  at  once  or,  more  often,  they  rotate  upon  it; 
and  in  the  latter  case,  looking  from  them  to  the  surface 
in  question,  in  a  counterclockwise  direction." 

In  contact  with  any  solid  object  Nereis  spermatozoa 
tend  to  carry  out  circus  movements  in  an  anticlockwise 
direction  when  fresh,  but  may  soon  come  to  rest.  This 
thigmotactic  reaction  appears  to  be  due  to  exaggeration 
of  the  rotation  component  of  the  ordinary  spiral  course; 
it  is  the  cause  of  aggregations  in  favoring  places,  such 
as  angles,  etc.  Such  contact  reactions  of  spermatozoa 
must  be  regarded  as  very  significant  for  fertilization. 

The  change  from  a  progressive  to  a  rotary  form  of 
locomotion  thus  appears  to  be  a  very  general  form  of 
reaction  of  spermatozoa  to  contact  stimuli.  This  may 
take  place  without  any  change  in  the  rate  of  activity 
or  of  metabolism  and  would  obviously  lead  to  aggre- 
gation of  spermatozoa  in  any  region  providing  a  stimulus 
which  acts  in  this  way. 

3.  In  Relation  to  Reaction  of  the  Medium;  H  and  OH  Ions 
a)  Activity  effects. — The  spermatozoa  of  many  ma- 
rine organisms  are  extremely  sensitive  to  changes  in 
the  normal  reaction  of  the  sea-water,  as  indicated  by 
their  activity.  In  the  case  of  Nereis  the  addition  of 
acids  (sulphuric,  hydrochloric,  nitric,  acetic)  to  the 
sea- water  caused  paralysis  at  w/ 1,000  and  decrease  of 
activity  up  to  n/^,000.  The  spermatozoa  of  sea  urchins 
are  not  so  sensitive  and  those  of  some  other  marine 
forms  much  less  so.  If  sea-water  is  saturated  with 
CO2  the  spermatozoa  of  all  forms  are  completely  para- 
lyzed, and  this  paralysis  will  last  in  the  case  of  Nereis 
until  the  charged  sea-water  is  diluted  over  a  hundred 
times;  in  i  per  cent  of  the  CO^  charged  sea-water  the 


lOO  PROBLEMS  OF  FERTILIZATION 

spermatozoa  show  no  movement,  and  it  is  not  until  a 
dilution  of  0.33  per  cent  is  reached  that  normal  activity 
is  possible.  Arbacia  sperm  exhibits  traces  of  movement 
at  2 . 5  per  cent  and  Chaetopterus  at  over  20  per  cent. 
There  is  thus  considerable  variation  in  this  respect 
(Lillie,  1913). 

Acids  decrease  the  activity  of  spermatozoa  up  to 
complete  paralysis,  and  at  higher  concentrations  cause 
death.  AlkaHes  have  in  general  the  reverse  action, 
increasing  activity  up  to  the  lethal  point.  The  death 
phenomena  of  sperm  suspensions  under  acids  and  alka- 
lies exhibit  a  striking  difference  in  gross  appearance, 
the  acid-killed  sperm  suspension  remaining  in  a  condi- 
tion of  dispersal,  whereas  in  the  alkali-  (KOH  or 
NaOH)  killed  sperm  suspensions  the  spermatozoa  are 
fused  in  strands  which  tend  to  anastomose;  a  similar 
gross  effect  upon  sperm  suspensions  is  also  produced  by 
the  salts  of  trivalent  metals  (Gray,  19 15). 

The  degree  of  activity  of  spermatozoa  is  a  function 
of  the  H  ion  concentration  of  the  medium,  other  things 
being  equal.  Kolliker  (1856),  Giinther  (1907),  Gray 
(19 1 5),  and  Cohn  (19 18)  have  all  recorded  similar  obser- 
vations. It  is  important  to  note  a  fact  emphasized  by 
Gray  that  spermatozoa  inactivated  by  acid  can  be 
reactivated  by  the  addition  of  alkaH  if  the  concentra- 
tion of  acid  used  in  the  experiment  has  not  been  too 
great;  the  inactivation  and  agglutination  produced  by 
excess  of  alkali,  on  the  other  hand,  is  irreversible. 

Cohn  (19 1 8)  has  shown  that  the  inactivation  by  H 
ions  has  very  important  consequences  for  the  func- 
tioning of  the  spermatozoa,  because  they  are  constantly 
giving  off  CO2  into  the  medium.     If  the  sperm  sus- 


PHYSIOLOGY  OF  THE  SPERMATOZOON 


lOI 


pension  be  above  a  very  low  degree  of  concentration 
the  H  ions  thus  liberated  soon  reach  a  sufficient  degree 
of  concentration  to  decrease  the  movements  of  the 
spermatozoa,  and  ultimately  stop  them  entirely.  The 
life  of  the  spermatozoa  is  thus  longer  in  more  concen- 
trated than  in  more  dilute  sperm  suspensions  because 
they  are  sooner  inactivated  by  their  own  CO^  and  the 
available  store  of  energy  is  not  used  up  so  rapidly. 
Spermatozoa  thus  inactivated  can  be  restored  to  full 
activity  merely  by  diluting  sufficiently  with  normal 
sea-water. 

The  effect  of  concentration  of  the  sperm  suspension 
on  the  duration  of  life  of  the  spermatozoa  has  been 
measured  by  Cohn,  who  used  the  fertilizing  power  as 
index  of  vitahty.  The  following  table  (after  Cohn) 
gives  the  results  of  one  such  experiment : 

The  Length  of  Life,  as  Measured  by  the  Fertilizing  Power,  of 
Sperm  Suspensions  of  Arhacia  of  Different 
Concentrations 


Age  oi 

Sperm 

Concentration  of  Sperm  Suspensions* 

Hours 

Minutes 

4  Per  Cent 

I  Per  Cent 

0.5  Per  Cent 

0.25  Per  Cent 

14 
23 

47 
71 

10 

40 

0 

55 
0 

100 

100 

100 

98 

85 

98 

98 

0 

0 

67 

15 
0 

10 

0 
0 

92 

*  Percentage  of  eggs  fertilized  at  age  of  sperm  in  same  horizontal  row. 


The  eggs  used  for  each  test  were  always  fresh. 
The  sperm  suspensions  were  made  up  at  the  same  time 
by  percentage  volume  of  sperm  to  sea-water;  they  were 
then  allowed  to  age,  as  shown  in  the  left-hand  column, 


I02  PROBLEMS  OF  FERTILIZATION 

and  each  was  tested  for  fertilization  power  at  the  ages 
given  by  adding  i  drop  of  sperm  to  5  drops  of  eggs  in 
10  c.c.  of  sea-water.  Such  sperm  suspensions  when 
perfectly  fresh  will  fertilize  100  per  cent  of  eggs  when 
so  used.  The  table  shows  that  the  fertilizing  power 
falls  off  with  age  in  inverse  proportion  to  concentration. 
This  is  in  inverse  relation  to  activity  and  to  CO2  and 
H  ion  concentration.  The  last  point  was  demonstrated 
by  measurements  of  the  rate  of  increase  of  H  ion  concen- 
tration in  sperm  suspensions  of  varying  concentration, 
which  runs  parallel  with  the  longevity. 

In  spite  of  the  more  rapid  rate  of  increase  in  H  ion 
concentration  with  sperm  concentration  Cohn  was  able 
to  show  that  the  amount  of  CO2  produced  per  unit  of 
sperm  is  ultimately  the  same  in  more  concentrated  and 
in  more  dilute  suspensions;  the  difference  is  merely  in 
the  rate  of  combustion,  which  is  more  rapid  in  propor- 
tion to  activity.  The  more  dilute  suspensions  use  up 
their  available  store  of  energy  more  rapidly,  the  more 
concentrated  suspensions  less  rapidly. 

h)  Aggregation  and  che^notaxis. — We  have  seen  that 
fresh  active  sperm  suspensions  of  Nereis  rapidly  form 
dense  aggregations  (p.  95,  Fig.  13)  in  masses.  Under 
a  low  power  of  the  microscope  each  mass  appears  like  a 
swarm  of  bees,  owing  to  intense  rotary  activity  of  the 
peripheral  spermatozoa.  But  those  in  the  interior  of 
the  dense  mass  must  be  quiescent.  The  reaction  is 
dependent  on  the  existence  of  a  certain  H  ion  concentra- 
tion of  the  sea-water,  for  if  the  sea-water  be  rendered 
hyperalkaline  the  aggregations  do  not  form,  however 
intense  the  activity  of  the  spermatozoa.  The  reaction 
is  in  fact  due  to  rapid  CO^  production  by  the  sper- 


PHYSIOLOGY  OF  THE  SPERMATOZOON  103 

matozoa,  which  tend  to  accumulate  in  any  region  of 
increased  CO2  tension.  Any  area  of  greater  concen- 
tration of  spermatozoa,  by  producing  more  CO^  than 
other  areas,  becomes  a  center  of  aggregation,  which, 
when  once  begun,  is  bound  to  proceed  to  the  limit  on 
account  of  increased  CO2  production.  The  reaction 
cannot  take  place  in  the  presence  of  excess  of  alkali, 
owing  to  neutralization  of  the  acid  as  fast  as  formed; 
and  a  CO2  tension  of  the  entire  medium  sufficient  to 
inhibit  movement  will  likewise  prevent  aggregation. 

It  is  obvious  that  the  aggregation  phenomenon  can 
be  explained  by  assuming  a  positive  orientation  of  the 
spermatozoa  in  a  CO2  gradient.  It  has  also  been  sug- 
gested (Cohn,  1 9 18)  that  it  is  due  merely  to  gradations 
of  activity,  and  therefore  presumably  of  the  range  of 
unimpeded  movement:  activity  decreases  with  rising 
CO2  tension;  the  spermatozoa  in  regions  of  higher  CO2 
therefore  tend  to  remain  there,  but  those  without, 
owing  to  their  wider  range  of  movement,  tend  to  move 
into  and  remain  within  such  regions. 

On  the  second  assumption  it  is  difficult  to  see  how 
it  happens  that  the  aggregations  become  so  dense. 
Neither  are  the  spermatozoa  motionless  in  such  aggre- 
gations as  required  by  this  theory,  but  they  can  be 
actually  observed  to  be  exceedingly  active  around  the 
periphery  of  the  aggregations,  and  their  movements 
are  almost  exclusively  those  of  rotation  such  as  are 
exhibited  in  the  thigmotactic  response. 

In  the  attempt  to  analyze  the  matter  farther,  the 
reaction  of  spermatozoa  to  solutions  of  CO,  in  sea-water 
was  tested  by  the  writer  by  the  PfeiTer  capillary-tube 
method,  which  consists  of  introducing  capillary  tubes 


I04  PROBLEMS  OF  FERTILIZATION 

filled  with  the  solution  to  be  tested  into  sperm  suspen- 
sions and  observing  the  reactions  of  the  spermatozoa 
to  the  open  end  of  the  tube.  Sea-water  was  charged 
to  saturation  with  CO2,  and  various  dilutions  of  such 
charged  sea-water  were  employed.  The  capillary-tube 
method,  however,  though  giving  positive  results,  proved 
inadequate,  on  account  of  the  very  slight  diffusion  from 
the  open  end  of  the  tube.  It  was  then  found  that  the 
injection  of  a  drop  of  the  solution  into  a  sperm  suspen- 
sion mounted  beneath  a  raised  cover  slip  gave  results 
at  least  ten  times  more  delicate.  Such  a  drop  is  con- 
fined above  and  below  by  the  glass  surfaces,  and  by 
diffusion  a  CO2  gradient  is  established  around  its 
margin.^ 

If  a  drop  of  a  I  per  cent  CO2  solution  in  sea-water^ 
be  introduced  into  a  fresh  milky  sperm  suspension  of 
Nereis  the  following  configuration  develops  in  a  few 
seconds  (Fig.  14a).  A  ring  of  densely  aggregated,  very 
active  spermatozoa  forms  near  the  margin  of  the  origi- 
nal drop,  and  a  similar  linear  aggregation  extends  from 

^  The  insistence  of  certain  investigators  on  the  Pfeffer  capillary- 
tube  method  (e.g.,  Loeb,  1916,  p.  93)  for  studying  chemotaxis  of  sper- 
matozoa is  difficult  to  understand.  The  effectiveness  of  the  Pfeffer 
method  depends  upon  diffusion  from  the  open  end  of  the  tube,  and  the 
gradient  is  therefore  estabhshed  for  the  greater  part  without  the  tube. 
The  amount  of  diffusion  depends  on  the  diameter  of  the  tube,  which 
is  rarely  stated  by  investigators;  the  walls  of  the  tube,  moreover, 
provide  a  source  of  thigmotactic  stimulation,  thus  interfering  with  the 
pure  chemotactic  reaction.  My  method  of  confining  a  drop  in  the  me- 
dium between  glass  surfaces  i  to  2  mm.  apart  provides  a  gradient  more 
surely  than  the  tube  method,  and  one  which  lasts  sufficiently  long  for 
all  practical  purposes.  It  is,  moreover,  much  simpler  and  more  easily 
controlled,  and  is  not  complicated  by  the  thigmotactic  factor. 

'  CO2  saturated  sea-water  diluted  a  hundred  times  with  normal 
sea-water. 


PHYSIOLOGY  OF  THE  SPERMATOZOON         105 

this  to  the  margin  of  the  slide  along  the  path  in  which 
the  pipette  was  introduced  and  withdrawn  (cf.  Fig. 
15).  The  ring  and  linear  aggregation  are  separated 
from  the  general  sperm  suspension  by  a  clear  space 
1.5  to  2  mm.  in  width.  No  such  reaction  takes  place 
with  reference  to  a  drop  of  normal  sea-water.     Greater 


Fig.  14. — Reaction  of  a  sperm  suspension  of  Nereis  to  a  drop  of 
I  per  cent  CO2  sea-water  (natural  size).  The  preparation  (a)  is  mounted 
on  a  slide  beneath  a  raised  cover  slip,  a  shows  the  form  of  the  reaction 
after  15  seconds;  b,  75  seconds;  c,  105  seconds;  d,  195  seconds.  In  d  the 
general  suspension  has  aggregated.  The  drop  to  the  right  in  a  is  a 
control  drop  of  sea-water.  Note  in  a  that  the  spermatozoa  also  with- 
draw from  the  margin  of  the  preparation,  thus  in  the  direction  of 
increasing  CO2  tension. 

dilutions  of  the  CO2  sea-water  will  act  positively  in 
the  case  of  fresh  sperm  suspensions.  The  ring  forms 
well  within  the  margin  of  the  drop  in  the  case  of  1/200 
dilution.  In  case  of  stronger  CO2  solutions  the  ring  is 
wider  and  tends  to  grow  at  the  periphery  as  the  CO2 
diffuses  outward.  In  general  the  aggregation  tends  to 
occur  at  a  place  in  the  CO2  gradient  near  the  point  of 
paralysis  of  the  spermatozoa. 


io6 


PROBLEMS  OF  FERTILIZATION 


It  is  clear  from  this  experiment  that  the  spermatozoa 
react  positively  in  a  CO2  gradient  where  the  tension  is 
above  a  certain  point;  the  aggregation  caused  by  a 
more  concentrated  drop  grows  because  the  diffusion  of 

CO2  from  the  center 
furnishes  a  widening 
ring  of  the  necessary 
concentration.  To 
furnish  a  gradient  the 
concentration  of  CO2 
must  exceed  that  in 
the  sperm  suspension, 
which  is  a  function 
of  its  density  and  age; 
on  the  other  hand,  a 
limit  is  set  to  the  dif- 
ferential which  fur- 
nishes the  reaction  by 
the  fact  that  a  con- 
centration of  about 
I    per    cent    of    the 


Fig.  15. — Diagram  of  the  reaction  of 
spermatozoa  of  Nereis  to  a  drop  of  i  per 
cent  CO2  in  sea-water.  The  dark  back- 
ground represents  the  sperm  suspension; 
the  thick  open  circle  and  streak  below,  the 
aggregation;  the  unbroken  line  represents 
the  original  boundary  of  the  COidrop;  the 
concentric  broken  lines  in  the  clear  zone 
represent  the  CO2  gradient. 


CO2  sea-water  para- 
lyzes the  spermato- 
zoa. The  gradient 
that  determines  the  reaction  must,  therefore,  exist  within 
very  narrow  limits. 

Such  aggregation  with  reference  to  CO2  is  obviously 
the  same  in  principle  as  the  aggregations  formed  spon- 
taneously in  sea-water.  Indeed,  aggregations  after- 
ward form  spontaneously  in  the  unaffected  part  of  such 
a  preparation.  The  spermatozoa  at  the  periphery  of 
the  ring  caused  by  CO2  are  in  much  more  rapid  move- 


PHYSIOLOGY  OF  THE  SPERMATOZOON  107 

ment  than  those  in  the  general  suspension,  where  they 
mutually  impede  one  another's  range  of  movement,  so 
that  it  seems  impossible  to  explain  the  aggregation  on 
the  assumption  that  it  is  due  to  CO2  paralysis.  This 
is  a  factor  in  the  result,  for  the  spermatozoa  that  gain 
the  center  of  the  drop  are  paralyzed,  but  paralysis 
explains  neither  the  initial  aggregation  nor  its  growth. 

The  behavior  of  the  spermatozoa  at  the  margin  of 
aggregations  is  curiously  like  the  thigmotactic  reaction 
previously  discussed ;  they  move  very  actively  in  circles 
of  increasingly  short  diameter  before  coming  to  rest.  At 
a  certain  point  in  the  CO2  gradient  the  circus  movements 
predominate  over  those  of  translation;  thus  spermatozoa 
reaching  this  point  in  the  gradient  are  practically  im- 
prisoned, although  active,  and  this  without  reference  to 
the  way  in  which  the  point  in  the  gradient  is  reached. 

If  the  clear  zone  of  a  fresh  preparation  be  carefully 
examined  it  can  be  seen  that  the  spermatozoa  are 
moving  across  it  in  streams  directly  toward  the  center; 
none  move  in  the  opposite  direction.  For  some  minutes 
this  steady  centripetal  migration  of  the  spermatozoa 
across  the  clear  zone  may  continue,  but  by  degrees  it 
ceases,  though  the  rotary  movement  of  the  spermatozoa 
at  the  margin  of  the  aggregation  continues  for  a  long  time. 
The  persistence  of  the  aggregation  is  thus  due  to  a 
behavior  change  which  is  the  same  as  that  given  in 
response  to  contact.  But  the  cause  of  the  aggregation 
lies  in  directive  movement  up  the  CO2  gradient  (chemo- 
taxis).  This  interpretation  supplements  the  account 
given  in  my  fifth  study  of  fertilization  (19 13). 

The  sperm  of  Nereis  exhibits  similar  behavior  with 
reference  to  other  acids,  thus  demonstrating  that  the 


io8  PROBLEMS  OF  FERTILIZATION 

H  ion  is  the  effective  factor.  The  spermatozoa  of 
Arbacia  exhibit  a  similar  but  much  less  pronounced 
behavior  and  only  to  considerably  higher  concentration 
of  CO2,  which  agrees  with  their  greater  tolerance  to 
H  ions. 

Alcohol  is  a  substance  which  paralyzes  the  sper- 
matozoa of  Nereis  at  5  per  cent,  and  greatly  decreases 
activity  at  2  per  cent.  However,  it  does  not  cause 
aggregations  similar  to  acids  when  tested  under  the 
same  conditions.  But  on  the  assumption  that  acid 
aggregations  are  due  to  progressive  paralysis,  i.e.,  that 
the  acid  acts  as  a  trap,  it  is  difficult  to  explain  why 
alcohol  does  not  act  similarly,  though  the  difference  is 
readily  understood  if  acids  cause  a  specific  form  of  be- 
havior and  alcohol  does  not.  Nor  do  the  spermatozoa 
of  Nereis  exhibit  any  aggregation  effect  with  reference 
to  drops  of  heated  or  cooled  sea-water,  which  have 
respectively  activating  and  inhibiting  effects. 

If  the  principle  of  chemotaxis  may  be  regarded  as 
applying  to  spermatozoa  in  the  foregoing  sense,  as 
I  believe  to  be  the  case,  it  is  obvious  that  this  form  of 
reaction  may  be  of  some  significance  in  bringing  the 
egg  and  spermatozoon  together  in  the  process  of  ferti- 
lization; we  shall  consider  this  subject  later,  but  the 
fact  of  capacity  for  chemotactic  aggregation  is  of  itself 
by  no  means  a  proof  that  such  aggregation  is  a  factor 
in  fertilization. 

4.  Reactions  to  Egg  Secretions 

The  reactions  of  spermatozoa  to  egg  secretions  of 
their  own  and  other  species  may  obviously  be  of  great 
significance  for  the  study  of  normal  and  hybrid  ferti- 
lization.    In  the  study  of  this  subject  it  is  important 


PHYSIOLOGY  OF  THE  SPERMATOZOON  109 

to  distinguish  between  egg  secretions  and  egg  extracts; 
we  shall  understand  by  secretions  the  normal  exudates 
of  the  egg  in  the  medium  in  which  insemination  takes 
place,  and  by  extracts  the  water-soluble  substances 
that  may  be  derived  from  the  egg  by  crushing  or  plas- 
molysis  in  an  aqueous  medium.  It  is  obvious  that  the 
extracts  will  contain  more  substances  than  the  secretion, 
and  different  kinds. 

In  the  first  place  it  is  known  that  the  eggs  of  marine 
animals  give  off  CO2  into  the  sea-water  before  fertili- 
zation. But  in  addition  to  CO2  other  substances  of 
complex  composition  are  given  off;  these  are  known 
mainly  by  their  effects  on  spermatozoa.  Four  such 
effects  may  be  noted  in  the  case  of  the  egg  secretions  of 
Arbacia  and  Nereis  and  some  other  forms,  viz.:  (i)  acti- 
vation: the  spermatozoa  are  stimulated  to  increased 
activity,  which  is  naturally  followed  sooner  or  later  by 
a  state  of  rest;  (2)  aggregation:  the  spermatozoa  are 
aggregated;  (3)  agglutination:,  the  spermatozoa  be- 
come stuck  together  in  temporary  clumps.  (The  first 
three  effects  are  noted  on  sperm  suspensions  of  the  same 
species.)  (4)  The  fourth  effect  is  noted  only  on  some, 
but  by  no  means  all,  foreign  sperm  suspensions;  it  is 
a  toxic  effect,  which  is  evidenced  by  irreversible  agglu- 
tination and  destruction  of  the  foreign  spermatozoa. 
The  spermatozoa  of  Nereis  for  instance  are  thus  de- 
stroyed by  the  egg  secretion  of  Arbacia,  but  the  effect 
is  not  reciprocal.  It  is  possible  that  the  last  effect  is 
produced,  not  by  an  egg  secretion  proper,  but  by  blood 
carried  over  into  the  egg  suspension. 

It  does  not  necessarily  follow  that  there  are  four 
substances  concerned  in  these  biological  reactions,  but 


no  PROBLEMS  OF  FERTILIZATION 

there  are  certainly  three,  and  we  shall  examine  the 
evidence  as  we  proceed. 

Methods  of  studying  egg  secretions:  The  present 
account  concerns  marine  animals  exclusively;  sea- water 
is  thus  the  medium  of  the  experiments.  Eggs  are 
allowed  to  stand  in  small  quantities  of  sea-water,  and 
in  a  short  time  the  sea-water  is  found  to  contain  the 
substances  concerned.  For  the  sake  of  brevity  we  shall 
call  this  sea-water,  egg  water.  The  indicator  is  a  sperm 
suspension  of  definite  concentration  in  sea-water,  which 
can  be  best  expressed  in  percentages  of  the  dry  sperm 
to  the  sea- water  of  the  suspension;  a  i  per  cent  sus- 
pension, i.e.,  I  part  of  dry  sperm  to  99  parts  sea-water, 
is  a  good  concentration.  The  most  generally  useful 
method  of  testing  the  reactions  is  to  place  some  drops 
of  the  sperm  suspension  on  a  slide  and  cover  it  with  a 
long  cover  slip  supported  by  glass  rods  about  i  mm. 
in  diameter;  the  egg  water  to  be  tested  is  then  injected 
into  the  suspension  with  a  capillary  pipette  operated 
by  a  long  rubber  tube  held  in  the  mouth.  By  this 
method  one  can  observe  under  the  microscope  all  the 
types  of  reaction.  But  some  kinds  of  observations  are 
better  made  in  test  tubes  or  other  containers. 

Egg  extracts,  as  contrasted  with  egg  secretions,  are 
prepared  by  mechanically  breaking  down  the  eggs  in 
sea-water,  by  grinding  dried  eggs  in  sea-water,  or  by 
plasmolyzing  in  distilled  water.  In  the  latter  case  the  dis- 
tilled water  extracts  may  be  brought  to  the  composition 
of  normal  sea-water  by  addition  of  its  volume  of  sea- water 
evaporated  to  half  its  original  volume.  We  shall  distin- 
guish such  preparations  as  egg  extracts  from  the  egg  water 
containing  merely  the  normal  secretions  of  the  eggs. 


PHYSIOLOGY  OF  THE  SPERMATOZOOxN 


III 


a)  Activity  ejfects. — The  spermatozoa  of  some  ani- 
mals, e.g.,  Nereis  and  Arbacia,  are  normally  active  in 
sea-water,  and  the  specific  egg  water  causes  no  noticeable 
acceleration  of  their  rate  of  movement  under  optimum 
conditions.  Others  again  are  very  inactive,  but  may  be 
aroused  to  intense  activity  by  the  specific  egg  water  or 
egg  extract.  This  is  true  of  the  spermatozoa  of  Asterias, 
for  instance,  during  the  summer  season  at  Woods  Hole. 
But  an  activating  substance  is  probably  present  even  in 
the  case  of  species  like  Arbacia.  Loeb  points  out  that 
the  spermatozoa  of  both  sea  urchins  and  starfish  are 
immobile  in  a  neutral  n/2  NaCl  solution  in  which  they 
will  continue  to  live  for  days,  but  the  addition  of  specific 
eggs  will  in  each  case  cause  immediate  and  often  intense 
activity.  There  is  a  certain  amount  of  specificity  in 
these  eft'ects,  as  is  shown  by  the  following  table,  taken 
from  Loeb  (1915): 

Specificity  of  xA.ctivation  of  Sperm  by  Eggs 


Asterias  S 

Asteritia  S 

A  rbacia  frati- 
ciscanus s 

Strongylocentro- 
tus purpuraius  S 

Asterias  ? 
(immature) 

Immediately 

No  activa-. 

Moder- 

Slight effect 

very  motile 

tion 

ately 

in  immedi- 

active 

ate  contact 
with  egg 

Aster ina  $ 
(immature) 

Not  motile 

Violent  ac- 
tivity 

Violent 
activity 

Slight  effect 
only   near 
the  egg 

Arbacia 
fraticiscanus  ? 
(mature) 

Slightly 
motile 

No  motility 

Immedi- 
ately 
active 

Immediately 
motile 

Strongylocentrotus 

Slightly 

Slight  effect  in 

Immedi- 

Immediately 

purpuraius  V 
(mature) 

motile   after 

immediate 

ately 

active 

some  time 

contact  with 
the  eggs 

active 

Thus  the  two  kinds  of  starfish  spermatozoa  seem  to 
exhibit    considerable    specificity    in    their    activation, 


112  PROBLEMS  OF  FERTILIZATION 

whereas  the  sea  urchin  spermatozoa  do  not  to  the  same 
extent.  The  latter  are,  however,  to  be  regarded  as 
always  nearer  the  threshold  of  activity.  Loeb  points 
out  that  the  activating  substance  is  different  from  the 
agglutinating  substance,  because  after  removal  of  the 
latter  the  activating  substance  is  still  found.' 

The  relationship  between  activity  of  the  spermatozoa 
and  presence  of  egg  secretions  may  obviously  be  a  sig- 
nificant factor  in  fertilization,  because  it  is  probable  that 
completely  immotile  spermatozoa  will  not  fertilize. 

b)  Aggregation  and  agglutination. — If  a  drop  of 
Arbacia  egg  water  be  injected  into  a  sperm  suspension 
of  the  same  species  beneath  a  raised  cover  slip  a  very 
violent  reaction  may  be  observed  under  a  low  power 
of  the  microscope.  In  the  first  second  the  spermatozoa 
within  the  drop  are  aroused  to  intense  activity  and  form 
small  agglutinated  masses;  these  then  fuse  with  the 
greatest  rapidity  to  form  larger  agglutination  masses  for 
a  period  of  three  to  five  seconds,  after  which  no  more 
fusion  of  masses  takes  place.  While  this  has  been 
going  on  in  the  interior  of  the  drop  a  ring  has  formed 
at  the  margin,  and  a  clear  zone  arises  external  to  it. 
The  ring  is  at  first  continuous,  but  it  ruptures  in  nu- 
merous places  in  two  or  three  seconds,  and  each  segment 
contracts  quickly  to  an  agglutinated  mass.  Such 
masses,  whatever  their  original  form,  quickly  contract 
into  spheres.  In  a  period  of  time  varying  from  a  few 
seconds  to  a  few  minutes,  depending  on  the  concentration 
of  the  egg  water,  the  agglutination  disappears.     The 

^His  statment  that  "Lillie  seems  to  take  it  for  granted  that  the 
substance  of  the  egg  which  causes  sperm  agglutination  is  identical  with 
the  substance  which  stimulates  the  spermatozoa  into  greater  activity" 
rests  upon  no  such  statement  of  mine. 


PHYSIOLOGY  OF  THE  SPERMATOZOON         113 

spermatozoa  are  then  for  a  time  relatively  immobile. 
The  same  observations  were  also  made  on  Nereis.  Loeb 
(19 14)  has  reported  the  occurrence  of  the  same  phe- 
nomenon in  various  species  of  sea  urchins,  Glaser  (19 14) 
in  Asterias,  and  Just  (19 18)  in  Echinarachnius . 

The  drop  of  egg  water,  two  to  four  millimeters  in 
diameter,  may  be  regarded  as  equivalent,  in  a  chemical 
sense,  to  a  much  magnified  Qgg,  and  the  reactions  of 
the  spermatozoa  to  it  as  similar  to  reactions  to  the 
actual  egg,  with  this  exception,  that  the  solid  surface  is 
lacking.  We  see  here  three  kinds  of  effects  of  the  spe- 
cific egg  water,  viz.,  activation,  aggregation,  and  aggluti- 
nation.' The  aggregation  effect  evidenced  by  the  ring 
and  clear  zone  with  reference  to  the  introduced  drop  is 
entirely  similar  to  the  aggregation  of  spermatozoa  of 
Nereis  produced  by  CO2  and  other  acids.  In  the  case 
of  the  Qgg  water  it  is  complicated  by  sunultaneous 
agglutination;  but  it  is  possible  to  eliminate  the  agglu- 
tination effect  (LilHe,  19 13)  by  neutrahzing  the  agglu- 
tinating substance  and  to  leave  the  aggregating  effect 

^  The  term  "agglutination"  will  be  used  exclusively  for  this  revers- 
ible phenomenon  of  adhesion  of  living  cells  for  a  longer  or  shorter 
time.  Agglutination  in  this  sense  has  no  effect  destructive  of  the  Hfe 
of  the  spermatozoon,  toxic  or  otherwise.  This  seems  to  the  author  a 
correct  biological  use  of  the  term.  Certain  other  substances,  such  as 
KOH,  NaOH,  or  certain  fluids  of  other  species,  cause  an  irreversible 
sticking  together  of  spermatozoa,  which  is  obviously  a  dififerent  phenom- 
enon biologically  and  is  usually  destructive.  This  is  to  be  distinguished 
sharply  from  biological  agglutination.  Confusion  is  likely  to  arise  in 
the  use  of  the  term,  because  Loeb,  for  instance,  has  called  the  caustic 
alkali  effect  "real  sperm  agglutination"  (1914,  pp.  126-27).  He  is 
here  using  the  term  in  its  etymological  significance.  The  effect  of 
caustic  alkalies  is  also  strikingly  different  in  appearance;  anastomosing 
cords  of  sperm  are  formed  constituting  a  network;  the  strands  never 
contract  into  spheres  as  in  agglutinated  sperm;  moreover,  the  sper- 
matozoa are  motionless  and  evidently  dead. 


114  PROBLEMS  OF  FERTILIZATION 

intact.^  In  this  case  the  ring  of  spermatozoa  remains 
unagglutinated.  Thus  the  aggregating  and  agglutinating 
substances  are  distinct.  We  have  previously  discussed 
the  significance  of  the  ring  formation  with  external 
clear  zone  with  reference  to  acids  in  the  case  of  Nereis 
and  arrived  at  the  conclusion  that  this  is  a  true  chemo- 
tactic  reaction  of  spermatozoa;  the  same  conclusion 
must  hold  for  the  egg  water  of  Arbacia,  which  thus 
contains  a  substance  derived  from  the  eggs  which 
determines  aggregation  of  spermatozoa. 

Egg  substances  that  thus  activate  and  direct  the 
specific  spermatozoa  and  render  them  adhesive  are  well 
adapted  to  favor  the  fertilization  reaction  which  we 
shall  consider  later. 

Doubt  has  been  expressed  as  to  the  presence  of  a 
chemotactic  agent  in  egg  secretions  of  animals.  Buller 
(1902),  who  investigated  the  subject  in  various  species 
of  sea  urchins,  using  the  tube  method  of  Pfeffer,  ob- 
tained only  negative  results.  He  states  that  they  went 
in  and  out  of  the  tubes  containing  egg  water  with  indif- 
ference, and  he  failed  to  discover  any  other  substance 
to  which  they  would  give  a  chemotactic  response  in 
the  sense  of  entering  the  solution  in  the  tubes  and 
remaining  there.  J.  de  Meyer  (191 1),  later  using  pre- 
cisely the  same  methods,  often  obtained  plugs  of  sper- 
matozoa several  millimeters  long  in  tubes  containing  egg 
water.     He  thus  disagrees  entirely  with  Buller;   but  as 

^  While  this  book  was  in  press  there  appeared  Forced  Movements, 
Tropisms  and  Animal  Conduct  by  Jacques  Loeb,  in  which  he  again 
returns  to  the  attack  against  chemotropism  of  spermatozoa.  His  some- 
what inaccurate  account  of  the  writer's  views  and  experiments  lends 
some  plausibility  to  criticisms  which  have  no  real  foundation;  the  funda- 
mental problem  involved  receives  no  real  consideration. 


PHYSIOLOGY  OF  THE  SPERMATOZOON  1 1 5 

neither  author  gives  any  quantitative  data  it  is  dif- 
ficult to  find  the  cause  for  the  disagreement.  As  I 
have  pointed  out  before,  the  tube  method  is  crude  as 
compared  to  the  injected-drop  method,  being  ten  to 
twenty  times  less  delicate.  A  negative  result  cannot 
be  trusted  for  this  reason,  and  a  positive  result  is  not 
necessarily  due  to  chemotactic  orientation,  for  sper- 
matozoa once  in  such  a  tube,  even  by  chance,  might  be 
imprisoned  there  by  any  paralyzing  effect  of  the 
contents. 

The  agglutination  effect  is  a  very  definite  and  char- 
acteristic reaction,  differing  from  mere  aggregation  in 
the  following  particulars:  in  the  latter  the  spermatozoa 
are  merely  loosely  associated,  and  sHght  agitation  is 
sufficient  to  scatter  them;  in  the  agglutinated  masses 
the  spermatozoa  are  stuck  together  and  are  not  sepa- 
rated by  shaking.  In  the  case  of  Nereis,  where  the 
agglutination  is  firmer  than  in  Arbacia,  the  masses  may 
be  broken  up  into  smaller  coherent  masses  by  needles 
or  preserved  intact  in  killing  fluids.  The  agglutinating 
substance  also  produces  its  characteristic  effect  when 
shaken  up  and  evenly  distributed  in  a  vial  of  sperm 
suspension,  but  an  aggregative  substance  cannot  of 
course  exert  a  chemotactic  effect  in  the  absence  of  a 
gradient.  The  agglutination  reaction  is  also  spontane- 
ously reversible,  unlike  aggregation;  moreover,  it  can- 
not be  repeated  if  the  reaction  is  complete,  owing  to 
complete  filiation  of  the  agglutinable  substance  born  by 
the  spermatozoon.  Although  at  one  time  Loeb  (1914) 
held  that  the  agglutination  was  probably  a  ''tropistic 
phenomenon,"  he  has  since  abandoned  this  view  (19 15, 
P-  275).     The  agglutinated  spermatozoa  are  living  and 


ii6  PROBLEMS  OF  FERTILIZATION 

apparently  in  nowise  injured,  in  which  respect  iso- 
agglutination  differs  from  the  frequent  toxic  effects  of 
foreign  egg  secretions,  which  may  cause  permanent 
adhesion  of  spermatozoa  in  masses. 

The  phenomenon  of  agglutination  of  sperm  suspen- 
sions by  egg  water  probably  does  not  occur  in  all 
animals.  Thus  I  have  been  unable  to  observe  it  in  the 
starfish,  though  Glaser  (19 14)  reports  its  occurrence  in 
this  form.  Absence  of  sperm  agglutination,  however, 
is  not  evidence  for  absence  of  a  comparable  secretion 
of  such  ova,  for  the  adhesion  of  the  spermatozoa  is 
evidently  a  result  of  a  surface  physical  change  of  sper- 
matozoa which  may  be  less  in  some  forms  than  in 
others.  Thus  while  in  some  species  the  spermatozoa 
are  efficient  indicators  for  the  substance,  they  need  not 
be  so  in  all.  This  does  not,  however,  in  the  least  de- 
tract from  the  usefulness  of  the  indicator  when  present. 

Loeb  points  out  that  sperm  suspensions  paralyzed 
by  KCN  do  not  agglutinate  in  specific  egg  water;  thus 
motility  of  the  spermatozoon  is  necessary  for  the  reac- 
tion; this  is  readily  understood  on  the  principle  that 
energy  of  impact  is  necessary  for  adhesion.  Loeb  also 
states  that  the  duration  of  the  reaction  is  dependent  to 
some  extent  on  the  alkalinity  of  the  medium.  "The 
more  alkaline  the  latter  the  more  rapidly  the  cluster 
scatters.  The  presence  of  a  salt  with  a  bivalent  metal, 
especially  Ca,  seems  necessary  for  the  cluster  for- 
mation." 

The  agglutination  reaction  may  be  studied  in  a 
quantitative  way.  The  reaction  is  reversible,  as  we  have 
seen;  with  high  concentration  of  the  agglutinating 
substance  it  may  be  several  minutes  before  the  aggluti- 


PHYSIOLOGY  OF  THE  SPERMATOZOON         1 1 7 

nated  masses  break  up  and  reversal  is  completed;  with 
low  concentrations,  on  the  other  hand,  the  agglutinated 
masses  are  smaller  and  their  disintegration  is  correspond- 
ingly more  rapid.  It  is  therefore  possible  to  establish 
a  unit  concentration  of  the  agglutinating  substance 
defined  as  the  greatest  dilution  at  which  an  unmistak- 
able reaction  is  given.  Such  a  reaction  lasts  only  four 
or  five  seconds,  and  the  agglutinated  masses  are  too 
small  to  be  seen  with  the  unaided  eye.  Any  given  egg 
water  may  therefore  be  rated  by  the  amount  of  dilution 
required  for  reduction  to  unit  strength,  as  containing 
10  or  100,  or  6,400,  etc.,  agglutinating  units.  The  highest 
concentration  obtained  in  my  experiments  on  Arbacia 
was  12,800  units.  Just  (19 19)  has  obtained  an  equally 
high  concentration  in  Echinarachnius  egg  water. 

c)  Properties  of  the  agglutinating  substance. — We 
may  now  examine  some  of  the  properties  of  the  agglu- 
tinating substance:  first,  its  biological  properties; 
sejcond,  its  physical  and  chemical  properties. 

Biological  properties:  Apart  from  the  data  already 
considered  we  may  note  that  the  eggs  alone  produce 
this  substance;  it  is  not  contained  in  the  blood  (peri- 
visceral fluid),  even  of  mature  females,  or  in  extracts 
of  any  other  tissues.  We  thus  have  a  specific  relation 
between  egg  and  spermatozoon  that  does  not  obtain 
between  any  other  tissues  and  the  spermatozoon.  The 
substance  is  tissue  specific. 

It  is  produced  by  mature  eggs  alone  and  ceases  to 
be  produced  by  fertilized  eggs.  Thus  its  production 
period  coincides  exactly  with  the  fertilizable  period  of 
the  ovum.  The  greatest  care  has  been  taken  to  deter- 
mine  this   point;     no   quantity   of   ovarian   substance 


iiS  •     PROBLEMS  OF  FERTILIZATION 

containing  only  immature  eggs  yields  even  a  trace  of 
sperm-agglutinating  substance;  and  after  the  eggs  are 
once  fertilized  and  the  jelly,  which  is  soaked  with  the 
substance,  is  removed  from  around  the  eggs  no  trace  of 
this  substance  is  ever  to  be  detected  from  these  eggs 
(cf.  also  Just  on  Echinarachnius,  191 9). 

The  agglutinating  substance  is  secreted  by  ferti- 
lizable  eggs  of  Arhacia  as  long  as  they  remain  in  a 
fertilizable  condition.  However,  the  jelly  membrane 
of  each  ^gg  is  saturated  by  the  substance,  as  is  readily 
shown  by  killing  the  eggs  by  heat  until  they  are 
thoroughly  coagulated,  when  the  jelly  still  continues  to 
give  off  the  substance  in  large  quantities  into  the  sea- 
water.  Loeb  (1914)  uidlxitdim&thditmStrongylocentrotiiS 
purpuratus  of  California,  eggs  deprived  of  jelly  lose  com- 
pletely and  permanently  the  power  of  agglutinating 
the  sperm  of  its  own  species;  that  the  jelly  alone  con- 
tains the  agglutinating  substance.  This  is  not  the 
case  in  Arhacia,  for  after  the  unfertilized  eggs  have 
been  deprived  of  jelly,  either  by  shaking  or  by  HCl, 
and  washed  several  times  to  remove  any  last  traces 
of  jelly,  they  still  continue  to  produce  the  agglutina- 
ting substance.  Thus  I  could  show  that,  whereas  the 
acid  solvent  which  removed  the  jelly  from  a  given 
lot  of  eggs  contained  only  400  agglutinating  units, 
after  a  series  of  washings  that  represented  a  dilution 
of  the  solvent  remaining  with  the  eggs  of  12,700,800 
times,  the  last  washing  agglutinated  the  spermatozoa. 
In  other  experiments  this  process  was  carried  much 
farther. 

In  a  considerable  number  of  experiments,  not  only 
by  myself  (1913,  1914),  but  also  by  C.  R.  Moore  (1917), 


PHYSIOLOGY  OF  THE  SPERMATOZOON         119 

the  capacity  of  Arbacia  eggs  to  continue  the  production 
of  the  agglutinating  substance  for  a  long  period  of  time 
after  removal  of  the  jelly  has  been  demonstrated. 
Glaser  (1914)  obtained  the  same  result.  To  obtain 
clear  evidence  of  the  reaction  from  such  eggs  the  obser- 
vations must  be  made  under  the  microscope,  preferably 
by  the  raised  cover-slip  method,  immediately  after  addi- 
tion of  the  egg  water,  for  the  reaction  lasts  only  from 
five  to  fifteen  seconds  and  the  agglutinations  are  micro- 
scopic in  size.  It  would  appear  probable  from  Loeb's 
account  that  he  used  only  macroscopic  methods,  and 
this  may  be  the  reason  for  his  negative  statement 
concerning  Strongylocentrotus. 

The  case  of  immature  eggs  also  shows  that  the 
agglutinating  substance  is  distinct  from  the  jelly,  be- 
cause these  eggs  are  already  provided  with  jelly,  but 
egg  water  from  them  contains  no  agglutinating  sub- 
stance. It  would  therefore  appear  that  this  substance 
begins  to  be  produced  during  the  process  of  maturation 
of  the  egg  and  is  discharged  from  the  egg  into  the  jelly, 
which  becomes  saturated  with  it,  and  will  therefore 
continue  to  yield  it  up  to  sea-water  even  when  sepa- 
rated from  the  egg.  These  results  are  completely 
confirmed  by  Just  (19 19)  for  Echinarachnius . 

The  agglutination  is  between  the  heads  of  the  sper- 
matozoa, which  obviously  become  adhesive  as  a  result 
of  action  of  the  egg  water;  the  tails  of  the  spermatozoa 
are  apparently  unaffected;  the  adhesive  change,  how- 
ever, soon  passes  away,  hence  the  subsequent  reversal 
of  agglutination.  The  loss  of  adhesive  properties  may 
be  due  to  solution  of  the  adhesive  substance  in  the 
sea-water  or  to  a  physical  change  in   the  substance. 


120  PROBLEMS  OF  FERTILIZATION 

In  the  case  of  Nereis  it  could  be  seen  that  in  aggluti- 
nated masses  the  heads  of  many  of  the  spermatozoa 
are  swollen  into  spherical  form  and  have  lost  their  nor- 
mal strong  refringibility;  in  such  a  case  they  are  usually 
motionless  and,  when  not  fused  with  one  another, 
appear  to  be  glued  to  the  slide  or  cover  slip. 

De  Meyer  (191 1)  observed  that  egg  extracts  of 
Echinus,  which  contain  certainly  other  substances  than 
the  secretions  of  uninjured  eggs,  cause  a  strong  swelling 
of  the  head  of  the  spermatozoon,  including  the  nucleus, 
and  other  transformations  depending  on  the  strength 
of  the  extract  and  the  duration  of  its  action;  the  swell- 
ing may  increase  the  diameter  as  much  as  eight  times. 
The  middle  piece  also  swells  and  may  divide.  The 
spermatozoa  thus  come  to  resemble  small  cells.  Thus 
he  states  that  in  sea-water  extract  of  eggs  the  spermat- 
ozoa undergo  some  of  the  changes  which  occur  within 
the  normally  fertilized  tgg. 

Chemical  and  physical  properties:  The  aggluti- 
nating substance  is  colorless;  it  will  not  pass  through  a 
Berkefeld  filter,  but  passes  readily  through  special  hard- 
ened filter  paper;  it  is  non-dialyzable;  it  is  extremely 
heat-resistant,  being  destroyed  only  slowly  at  the 
boiling-point;  it  may  be  kept  in  sea-water  for  months, 
though  it  slowly  disintegrates.  It  is  obviously  colloidal 
in  its  character,  but  Glaser  (1914)  has  determined  that 
it  does  not  give  the  usual  protein  tests :  Millon's  reagent 
gave  a  white  precipitate  with  no  color  changes  on 
boihng;  the  biuret  test  was  negative;  HNO3  gave  no 
ring,  but  a  faint  cloudiness;  the  xanthoproteic  test 
gave  no  precipitate,  but  the  solution  turned  distinctly 
yellow;    the  Adamkiewicz  test  was  negative;    Fehling 


PHYSIOLOGY  OF  THE  SPERiMATOZOON  1 2 1 

gave  no  reduction;  bisubnitrate  gave  no  reduction. 
Richards  and  Woodward  (191 5)  point  out  that  the 
efficiency  of  the  agglutinin,  like  pepsin,  varies  with  the 
square  root  of  the  concentration.  If  the  efficiency  is 
measured  by  the  number  of  seconds  the  spermatozoa 
remain  agglutinated,  and  the  concentration  is  measured 
by  units  of  strength,  a  curve  results  of  approximately  the 
formula  y''  =  iix,  where  y  represents  the  efficiency  and 
X  the  concentration  (Richards  and  Woodward,  191 5). 
The  same  authors  also  state  that  X-radiation  affects 
solutions  of  the  agglutinating  substance  in  the  same 
sense  as  ferments,  accelerating  in  a  short  exposure 
(about  two  minutes),  non-effective  in  a  five-minute  expo- 
sure, and  inhibitive  in  a  longer  exposure.  It  thus 
possesses  some  ferment  analogies. 

A  completely  agglutinated  sperm  suspension  in  which 
reversal  has  occurred  is  not  capable  of  reagglutination 
by  the  addition  of  more  of  the  agglutinating  substance, 
and  the  substance  disappears  from  an  agglutinated 
suspension  when  not  present  in  excess.  The  agglu- 
tination has  therefore  some  of  the  usual  characters  of 
a  chemical  reaction.  Glaser  (19 14)  has  also  made  the 
same  determination.  Schiicking  (1903)  also  holds  to 
a  union  of  the  agglutinating  substance  of  the  egg 
with  the  agglutinable  substance  of  the  sperm  for  similar 
reasons.  The  writer  (1914)  has  determined  that  i  c.c. 
of  3  per  cent  sperm  suspension  of  Arhacia  will  fix  about 
64  units  of  the  agglutinating  substance,  i.e.,  i  c.c.  of 
Qgg  water  of  64-unit  agglutinating  strength.  In  more 
specific  terms,  if  i  c.c.  of  3  per  cent  sperm  be  added  to 
I  c.c.  of  64-unit  egg  water  and  the  spermatozoa  be 
precipitated   after   agglutination   by   centrifuging,    the 


122  PROBLEMS  OF  FERTILIZATION 

agglutinating  substance  is  found  to  be  absent  from  the 
supernatant  fluid.  If  a  lesser  quantity  of  sperm  be 
employed  some  agglutinating  substance  will  remain  free. 
Whether  we  are  dealing  here  with  a  true  chemical  union, 
or  merely  with  a  process  of  adsorption  is  not  yet 
determined. 

If  sperm  suspensions  are  allowed  to  stand  for  some 
hours  they  reach  a  condition  where  they  will  aggluti- 
nate only  with  strong  solutions.  If  the  binding  power 
is  then  tested  it  is  found  that  they  fix  as  much  of  the 
agglutinating  substance  as  when  fresh.  The  fixing 
power  of  the  sperm  is  thus  entirely  independent  of  its 
capacity  for  being  agglutinated,  which  depends  upon 
the  freshness  of  the  spermatozoa.  The  binding  capa- 
city would  appear,  therefore,  to  depend  upon  the  pres- 
ence of  a  certain  Substance  borne  by  the  spermatozoon 
which  we  may  call  the  agglutinable  substance.  We 
may  suppose  that  in  stale  sperm  suspensions  this  sub- 
stance may  be  cast  off  and  lie  free  in  the  medium. 
But  this  has  not  been  actually  determined. 

5 .  "  Hetero-A  gglutination ' '  and  Specificity 

The  egg  waters  of  Arbacia  and  of  Nereis  possess 
substances  each  agglutinating  for  its  own  sperm.  If 
we  test  the  Nereis  egg  water  on  Arbacia  sperm  we  find 
that  it  is  entirely  negative;  if  on  the  other  hand  we 
test  the  Arbacia  egg  water  on  Nereis  sperm  the  latter 
is  apparently  strongly  agglutinated.  At  first  sight  it 
would  seem  that  there  is  specificity  in  the  one  direction 
but  not  in  the  other  with  reference  to  the  agglutinating 
substances.  If,  however,  we  examine  the  effect  of  the 
Arbacia  egg  water  on  Nereis  sperm  more  carefully  we 
find  that  the  reaction  bears  quite  a  different  character 


PHYSIOLOGY  OF  THE  SPERMATOZOON  123 

from  either  iso-agglutination.  The  adhesion  of  the 
spermatozoa  is  permanent,  not  reversible,  and  if  the 
egg  water  be  strong  the  spermatozoa  are  killed.  More- 
over, the  blood  of  Arhacia  produces  the  same  effect  on 
Nereis  sperm,  as  its  egg  water,  whereas  it  is  an  indif- 
ferent medium  for  the  specific  sperm.  The  conclusion 
is  therefore  suggested  that  the  "hetero-agglutinin"  and 
the  ''iso-agglutinin"  of  Arbacia  egg  water  are  dift'erent 
substances. 

This  can  in  fact  be  demonstrated  in  more  than  one 
way.  Thus  an  Arhacia  Qgg  water  that  originally  acted 
both  on  Arhacia  and  Nereis  sperms  was  found  to  have 
lost  all  effect  on  Nereis  sperm  after  seventeen  days, 
whereas  it  retained  undiminished  its  agglutinating 
effect  on  its  own  sperm  (Lillie,  19 13).  The  non-specific 
substance  was  destroyed  by  the  chemical  changes  in 
the  egg  water,  but  the  specific  substance  remained. 
It  is  also  possible  to  precipitate  out  all  of  the  Nereis- 
active  substance  with  Nereis  sperm  and  leave  the  full 
complement  of  iso-agglutinating  substance.  Thus  a 
sample  of  Arhacia  egg  water  was  found  to  have  800- 
unit  iso-agglutinating  power;  it  had  also  a  powerful 
effect  on  Nereis  sperm  but  was  negative  at  one- 
sixteenth  dilution.  The  addition  of  two  drops  of  a  i 
per  cent  sperm  suspension  of  Nereis  to  i  c.c.  of  the 
egg  water  completely  neutralized  the  Nereis  active  sub- 
stance but  left  the  original  800-unit  strength  of  the  iso- 
agglutinating  substance.  The  sperm  of  a  teleost  was 
also  found  capable  of  neutralizing  the  hetero-active 
substance,  leaving  the  iso-agglutinating  substance  in- 
tact. The  hetero-active  substance  would  thus  appear 
to    be    rather    generally    toxic    to    foreign    sperm.     It 


124  PROBLEMS  OF  FERTILIZATION 

occurs,  as  we  have  seen,  in  the  blood  of  the  sea  urchin 
and  is  present  there  in  greater  concentration  than  in 
the  egg  water;  so  it  is  certainly  not  tissue  specific  as 
is  the  iso-agglutinin. 

It  is  therefore  certain  that  the  iso-agglutinins  of 
Arhacia  and  of  Nereis  are  without  action  on  the 
heterologous  sperm.  But  these  forms  belong  to  differ- 
ent phyla.  How  is  it  as  between  more  closely  related 
forms?  Just  (19 19)  has  found  that  Qgg  water  of  Arha- 
cia agglutinates  the  sperm  of  Arhacia  and  Echinarach- 
nius;  the  egg  water  of  the  latter  will  agglutinate  its 
own  sperm  but  not  that  of  Arhacia.  The  relations  are 
thus  similar  to  Arhacia  and  Nereis;  and  correspond- 
ingly Just  was  able  to  show  that  two  distinct  substances 
are  involved  in  the  Arhacia  Qgg  water.  Loeb  (19 14, 
p.  125)  notes  that  the  Qgg  water  of  the  sea  urchin 
Stfongylocentrotus  purpuratus  will  agglutinate  both  its 
own  sperm  and  that  of  S.  franciscanus  but  the  latter 
not  so  strongly;  the  egg  water  of  5.  franciscanus  will 
agglutinate  its  own  sperm  but  not  that  of  S.  purpuratus. 
Here  again  the  specificity  is  not  reciprocal,  and  this 
suggests  the  possibility  that  the  hetero-agglutinating 
substance  of  S.  franciscanus  may  be  a  distinct  substance 
from  the  iso-agglutinating  substance.  Loeb,  however, 
did  not  investigate  this  possibility. 

The  results  thus  far  obtained  are  too  meager  to 
permit  a  generalization  on  the  specificity  of  the  agglu- 
tination reaction.  There  is  a  considerable  degree  of 
specificity  in  such  reactions,  as  we  have  seen,  but  we  do 
not  yet  know  how  far  this  extends. 

Certain  observations  indicate  an  absence  of  agglu- 
tinating effect  of  egg  water  on  specific  sperm  of  certain 


PHYSIOLOGY  OF  THE  SPERMATOZOON         125 

species;  wc  do  not  indeed  know  how  widespread  the 
phenomenon  may  be.  Where  it  occurs  we  have  a 
definite  indicator  of  a  reaction;  the  failure  of  the  indi- 
cator in  certain  species  is  no  evidence  of  lack  of  such 
reaction,  for  the  fundamental  reaction  is  on  the  individ- 
ual spermatozoon;  the  agglutination  is  a  consequence 
of  secondary  conditions  such  as  the  concentration 
of  the  sperm  suspension,  the  rate  of  movement  of  the 
affected  cells,  and  the  composition  of  the  medium;  it 
may  also  be  that  the  reaction  in  certain  species  does 
not  involve  such  physical  alteration  of  the  sperm  proto- 
plasm as  to  permit  of  agglutination  to  one  another. 
We  must  therefore,  I  believe,  utilize  such  positive  indi- 
cations as  we  have  in  our  analysis,  leaving  the  negative 
cases  for  future  investigation. 

The  fertilization  reaction  proper  is  considered  in  chap- 
ter vii.  It  is  obvious  that  for  the  purposes  of  this  reac- 
tion, which  involves  adhesion  and  ultimate  fusion  of  the 
gametes,  activation  of  the  spermatozoon,  tendency  to 
collect  in  the  region  of  egg  secretions,  the  thigmotropic 
reaction,  and  the  .development  of  an  adhesive  surface 
by  action  of  the  agglutinating  substance  of  the  egg 
constitute  forms  of  behavior  exactly  suited  to  the  final 
accomplishment.  The  layer  of  jelly  that  invests  the 
ova  of  most  marine  forms  acts  mechanically  to  entangle 
the  spermatozoa,  and  it  also  concentrates  the  action 
of  the  egg  secretions,  with  which  it  is  heavily  charged. 

The  meeting  of  the  egg  and  the  spermatozoon  is 
to  be  regarded  neither  as  a  matter  of  random  activity 
of  the  spermatozoon  alone,  as  some  have  been  inclined 
to  regard  it,  nor  yet  exclusively  as  a  result  of  direct 
orientation  of   the  spermatozoon   toward   the   egg  by 


I  26  PROBLEMS  OF  FERTILIZATION 

chemotaxis,  as  others  have  supposed.  It  appears  really 
to  be  a  more  complex  event  in  which  the  various  forms 
of  behavior  of  the  spermatozoon  may  all  play  a  part, 
as  indicated. 

REFERENCES 
Ballowitz,  Emil. 

1890.  "  Untersuchungen  ueber  die  Structur  der  Sperma- 
tozoen,"  Zeitschr.  fiir  wiss.  Zool.     (See  pp.  392-93). 

BULLER,   A.    H. 

1902.     "Is  Chemotaxis  a  Factor  in  the  Fertilization  of  the 
Eggs  of  Animals?"     Quart.  Jour.  Micr.  Set.,  XLVI, 
145-76. 
CoHN,  Edwin  J. 

191 7.  "The  Relation  between  the  Hydrogen  Ion  Concen- 
tration of  Sperm  Suspensions  and  Their  Fertilizing 
Power,"  Anat.  Rec,  II,  530. 

1918.  "Studies  in  the  Physiology  of  Spermatozoa,"  Biol. 
Bull.,  XXXIV,  167-218.  ^ 

Dewitz,  J. 

1885.  "Ueber  die  Vereinigung  der  Spermatozoen  mit  dem 
Ei,"  Arch,  fiir  d.  ges.  Physiol.,  Band  37,  pp.  219-23. 

1886.  "Ueber  Gesetzmassigkeit  in  der  Ortsveranderung 
der  Spermatozoen  und  in  der  Vereinigung  derselben 
mit  dem  Ei,"  ibid.,  Band  2)^,  pp.  358-85. 

DuNGERN,  Emil  von. 

1902.  "Neue  Versuche  zur  Physiologie  der  Befruchtung," 
Zeitschr.  fiir  all  gem.  Physiol.,  Band  I,  pp.  34-55. 
See  also  Centralblatt  fiir  Physiol.,  1901. 

Gemmil,  James  J. 

1900.  "On  the  VitaHty  of  the  Ova  and  Sperm  of  Certain 
Animals,"    Jour,  of  Anat.  and  Physiol.,  XXXIV,  163. 

Glaser,  Otto. 

1914.  "A  Qualitative  Analysis  of  the  Egg  Secretions  and 
Extracts  of  Arhacia  and  Asterias,^'  Biol.  Bull.,  XXVI, 
367-86. 


PHYSIOLOGY  OF  THE  SPERMATOZOON  127 

Glaser,  Otto. 

1915.     ''Can  a  Single  Spermatozoon  Initiate  Development 
in  Arbaciar'     Biol.  Bull.,  XXVIII,  149-53. 
Gray,  James. 

191 5.  "Note  on  the  Relation  of  Spermatozoa  to  Electro- 
lytes and  Its  Bearing  on  the  Problem  of  Fertiliza- 
tion,"    Quart.  Jour.  Micr.  Sci.,  N.S.,  LXI,  119-26. 

GUNTHER,    GUSTAV. 

1907.  "Ueber  Spermiengifte,"  Arch,  fiir  d.  gcs.  Physiol., 
Band  118,  pp.  551-71. 

Just,  E.  E. 

1919.  "The  Fertilization  Reaction  in  Echinarachnius 
parma.  II,  The  Role  of  Fertilizin  in  Straight  and 
Cross-Fertilization,"  Biol.  Bull.,  XXXVI,  11-38. 

KOLLIKER,    A. 

1856.  " Physiologische' Studien  liber  die  Samenfliissigkeit," 
Zeitschr.  fiir  wiss.  Zool.,  Band  7,  pp.  201-73. 

LiLLiE,  Frank  R. 

1913.  "Studies  of  Fertilization.  V.  The  Behavior  of  the 
Spermatozoa  of  Nereis  and  Arhacia  with  Special 
Reference   to   Egg   Extractives,"   Jour.   Exp.   Zool., 

XIV,  515-74. 

1914.  "Studies  of  Fertilization.  VI.  The  Mechanism  of 
Fertilization  in  Arbacia,"  ibid.,  XVI,  523-90. 

191 5.  "Sperm  Agglutination  and  Fertilization,"  Biol. 
Bull,  XXVIII,  18-33. 

"Studies  of  Fertilization.  VII.  Analysis  of  Varia- 
tions in  the  Fertilizing  Power  of  Sperm  Suspensions 
of  Arabacia,"  ibid.,  pp.  229-51. 

LoEB,  Jacques. 

1914.  "Cluster  Formation  of  Spermatozoa  Caused  by 
Specific  Substances  from  Eggs,"  Jour.  Exp.  Zool., 
XVII,  123-40. 

191 5.  "On  the  Nature  of  the  Conditions  Which  Determine 
or  Prevent  the  Entrance  of  the  Spermatozoon  into 
the  Egg,"  Amer.  Naturalist,  XLIX,  257-85. 


128  PROBLEMS  OF  FERTILIZATION 

LoEB,  Jacques 

191 6.  The  Organism  as  a  Whole  from  a  Physicochemical 
Viewpoint.     New  York:   G.  P.  Putnam's  Sons. 

Low,  O. 

1902,  1903.     "Die  Chemotaxis  der  Spermatozoa  im  weibli- 

chen  Genitaltract,"  Sitzungsher.  d.  kdnigl.  Akad.  d. 
Wiss.,  Math.  Naturw.  Kl.,  Wien,  Band  111-12. 

Massart,  Jean. 

1888.  "  Sur  rirritabilite  des  spermatozoides  de  la  grenouille," 
Bull,  de  VAcad.  Roy.  des  Sci.  de  Belgique,  T.  15,  pp. 
750-54. 

1889,  "Sur  la  penetration  des  spermatozoides  dans  I'oeuf 
de  la  grenouille,"  ibid.,  T.  18,  pp.  215-20. 

De  Meyer,  J. 

191 1.  "Observations  et  experiences  relatives  a  Taction 
exercee  par  des  extracts  d'oeufs  et  d'autres  substances 
sur  les  spermatozoides,"  Arch,  de  bioL,  T.  26,  pp. 
65-101. 

Moore,  C.  R. 

191 7.  "On  the  Capacity  for  Fertilization  after  Initiation 
of  Development,"  Biol.  Bull.,  XXXIII,  258-95. 

Pfeffer,  W. 

1884.  "Locomotorische  Richtungsbewegungen  durch  chem- 
ische  Reize,"  Untersuchiingen  a.  d.  bot.  Inst,  zu 
Tubingen,  Band  i. 

Richards,  A.,  and  Woodward,  A.  E. 

191 5.  "Note  on  the  Effect  of  X-Radiation  on  Fertilizin," 
Biol.  Bull,  XXVm,  140-47. 

SCHUCKING,   A. 

1903.  "Zur  Physiologic  der  Befruchtung,  Parthenogenese 
und  Entwickelung,"  Arch,  fiir  d.  ges.  Physiol.,  Band 
97,  pp.  58-97. 

WiNSLOW,    G.   M. 

1903.  "Note  on  Circular  Swimming  of  Sand-Dollar  Sper- 
matozoa," Science,  N.S.,  XVII,  153. 


CHAPTER  V 

THE  PHYSIOLOGY  OF  FERTILIZATION 
I.    INTRODUCTION 

It  is  commonly  said  that  there  are  two  main  problems 
in  the  physiology  of  fertilization,  viz. :  the  initiation  of 
development,  or  activation,  and  biparental  inheritance; 
but  these  are  more  properly  results  of  fertilization.  In- 
deed so  long  as  we  regard  fertilization  primarily  as  a 
function  of  prospective  significance  in  the  life  of  the 
organism  we  shall  miss  the  more  specific  aspects  of  the 
process.  Once  fertilization  is  accomplished  development 
and  inheritance  may  be  left  to  look  after  themselves. 

Fertilization  must  be  regarded  as  a  reaction  possess- 
ing very  definite  biological  and  biochemical  characters. 
But  it  is  not  a  single  reaction  in  either  a  biological  or 
chemical  sense;  it  is  rather  a  series  of  reactions  which 
cannot  be  regarded  as  complete  until  full  capacity  for 
development  and  inheritance  is  attained  by  the  zygote. 
Fertilization  may  therefore  be  partial,  which  may  be 
indicated  by  early  cessation  of  the  developmental  pro- 
cess or  deficiency  of  developmental  energy.  It  is  obvi- 
ous that  partial  fertilization  will  furnish  an  important 
means  of  analysis. 

The  reactions  of  fertilization  form  an  irreversible 
series,  though  it  is  conceivable  that  some  of  them  taken 
singly  may  be  reversible.  Spermatozoa  have  no  effect  on 
already  fertilized  eggs  and  do  not  penetrate  into  them; 
this    condition    arises    very  early    in    the   fertilization 

129 


I30      ■  PROBLEMS  OF  FERTILIZATION 

reactions,  so  that  the  entrance  of  more  than  one  spermat- 
ozoon does  not  usually  occur.  With  reference  to  rever- 
sibility the  ovum  differs  from  most  cells.  A  nerve  cell  or 
a  gland  cell  or  a  muscle  cell  after  functioning  returns 
to  the  functional  condition  again;  but,  so  far  as  our 
evidence  goes,  this  is  not  true  of  the  egg  cell  or  of  the 
spermatozoon;  their  functioning  is  essentially  a  pro- 
gressive process,  the  cycle  of  which  occupies  an  entire 
generation.  From  this  point  of  view  the  lack  of 
reversibility  is  readily  understood. 

Fertilization  is  a  specific  reaction  in  the  sense  that 
it  occurs  usually  only  between  the  gametes  of  the  same 
species.  Hybridization  is  possible  within  certain  indef- 
inite limits,  which  do  not  follow  any  invariable  taxo- 
nomic  rule,  though  as  a  general  thing  it  is  regarded  us 
increasingly  improbable  with  remoteness  of  taxonomic 
relationship.  In  the  echinids  and  teleosts,  however, 
hybrid  fertilization  is  possible  even  between  different 
families  or  suborders.  Resistance  to  hybridization  may 
also  be  broken  down  by  certain  experimental  proce- 
dures considered  later.  On  the  other  hand  we  have 
the  special  phenomenon  of  self-infertility  in  certain 
hermaphrodites  (see  chap.  vi). 

Certain  eggs  are  normally  parthenogenetic,  and  in 
others  artificial  parthenogenesis  may  be  produced  by 
various  experimental  procedures.  It  is  obvious  that 
in  such  cases  the  activating  effects  of  fertilization  are 
produced,  and  that  the  analysis  of  parthenogenesis  must 
have  very  direct  bearings  on  this  problem  of  fertili- 
zation. We  shall  therefore  utilize  the  results  of  such 
studies  to  a  certain  extent,  but  only  as  an  aid  in  the 
analysis  of  fertilization  (see  chap.  vii). 


THE  PHYSIOLOGY  OF  FERTILIZATION  131 

It  is  usual  to  regard  penetration  of  the  spermatozoon 
as  synonymous  with  fertilization,  but  we  may  have 
fertilization  well  begun  after  mere  attachment  of  the 
spermatozoon  and  without  any  penetration,  as  in 
Nereis;  on  the  other  hand  we  may  have  penetration 
without  any  fertilization,  if  the  egg  is  not  in  the  proper 
condition.  A  complete  fertilization  reaction  involves 
penetration,  which  thus  furnishes  one  of  the  problems 
of  fertilization;    but  it  is  not  fertilization  in  itself. 

II.    THE   FERTILIZABLE    CONDITION   OF  THE  G.AMETES 

The  fertilization  reactions  are  possible  only  during  a 
definite  hmited  time  in  the  life  of  the  gametes.  This 
has  usually  been  attributed  to  rapid  aging  after  the 
fertilizable  condition  is  once  attained,  but  a  careful 
examination  of  the  loss  of  fertilizing  power  may  enable 
us  to  define  this  problem  more  exactly. 

I.  The  spermatozoon. — Spermatozoa  attain  fertilizing 
power  after  the  completion  of  histogenesis  and  the 
attainment  of  full  motility.  Unripe  spermatozoa  will 
not  fertilize.  After  ripening,  spermatozoa  retain  ferti- 
lizing power  for  variable  periods  depending  on  the 
species  and  on  the  conditions.  Within  the  gonads  or 
in  the  seminal  ducts  spermatozoa  are  motionless  and 
may  remain  in  good  condition  apparently  indefinitely 
until  ejaculated.  In  the  case  of  animals  with  internal 
fertilization  the  life  of  spermatozoa  within  the  genital 
tract  of  the  female  may  also  be  considerable.  In  birds 
(fowl)  spermatozoa  may  retain  power  for  two  or  three 
weeks.  In  most  mammals  the  period  is  shorter;  but 
in  bats,  for  instance,  copulation  occurs  in  the  fall,  and 
ovulation  and  fertilization  are  delayed  until  the  spring. 


132  PROBLEMS  OF  FERTILIZATION 

The  females  of  many  insects  possess  seminal  receptacles 
in  which  spermatozoa  received  from  the  male  may  be 
stored  up  for  long  periods;  in  the  case  of  ants  and  bees, 
for  instance,  spermatozoa  within  the  seminal  receptacles 
may  retain  their  fertilizing  power  for  years. 

The  actual  conditions  of  vitality  and  fertilizing 
power  can,  however,  best  be  studied  in  forms  with 
external  insemination,  especially  marine  forms.  Sea 
urchins  furnish  ideal  material  for  such  a  study.  In 
this  case  it  has  been  shown,  especially  by  the  studies  of 
E.  J.  Colin  (19 1 8),  that  the  vitaHty  and  fertilizing 
power  endure  in  inverse  ratio  to  activity.  Concen- 
trated sperm  suspensions  retain  their  vitality  longer 
than  more  dilute  suspensions  because  they  rapidly  pro- 
duce a  paralyzing  concentration  of  CO2  (see  p.  loi). 
Similarly,  any  substance  that  will  paralyze  the  spermat- 
ozoa without  killing  them,  such  as  dilute  acids,  KCN, 
etc. ,  will  prolong  the  period  of  fertilizing  power.  Cohn  has 
shown  that  Fuch's  (191 5)  result  on  increase  in  duration 
of  fertilizing  power  of  sperm  suspensions  in  specific  egg 
water  as  compared  with  similar  suspensions  in  sea-water 
is  due  to  the  paralyzing  effect  of  the  water  on  the  sperm. 

The  question  may  now  be  raised  whether  the  ferti- 
lizing power  of  sperm  suspensions  is  dependent  exclu- 
sively on  motility,  or  whether  the  presence  of  a  specific 
substance  borne  by  the  sperm  is  not  also  necessary? 
Such  a  substance  must  be  borne  superficially,  for  the 
fertilization  reaction  begins  immediately  after  attach- 
ment of  the  spermatozoon  to  the  egg.  Loeb  (1913, 
p.  227)  at  one  time  postulated  the  idea  that  the  sperm 
bears  a  lysin-like  substance  producing  superficial  cy- 
tolysis  of  the  egg,  which  he  regarded  as  the  first  step 


THE  PHYSIOLOGY  OF  FERTILIZATION  133 

in  initiation  of  development.  Such  a  substance  might 
conceivably  be  lost  by  the  spermatozoon  without  detri- 
ment to  its  vitality,  leaving  the  spermatozoon  in  a 
motile  condition  but  without  capacity  for  fertilization. 

The  conception  of  a  fertilizing  substance  borne  by 
the  spermatozoon  seems  to  be  a  necessary  one,  but  the 
conception  that  it  acts  on  the  egg  as  a  cytolytic  agent 
is  no  longer  maintained.  The  attempt  has  been  made 
by  a  number  of  investigators  (Winkler,  1900;  Gies, 
1901;  Robertson,  191 2)  to  extract  such  a  substance 
from  spermatozoa,  but  without  success  in  the  produc- 
tion of  a  solution  that  will  cause  development  of  the 
egg  of  the  same  species. 

T.  B.  Robertson  (191 2)  has  extracted,  by  a  very  com- 
plex process,  from  the  sperm  of  the  sea  urchin  an  acid- 
soluble  substance  which  caused  the  production  of 
atypical  membranes  on  sea  urchin  eggs,  especially 
where  the  action  was  reinforced  by  previous  treat- 
ment of  the  eggs  with  f  NSrCL.  No  development 
took  place.  It  seems  doubtful  to  the  writer  that  this 
represents  the  specific  membrane-producing  substance 
of  the  spermatozoon,  as  Robertson  maintains.  The  sub- 
stance proved  markedly  poisonous  to  the  eggs,  an  effect 
that  was  not  removed  by  treatment  with  h>^ertonic  sea- 
water.  It  seems  possible  that  some  cleavage  product  of 
the  sperm  proteins  was  concerned.  As  the  results  of 
other  authors  (Gies,  etc.)  have  been  negative,  and,  more- 
over, as  Loeb  has  treated  the  subject  quite  fully  in  his 
book  on  Artificial  Parthenogenesis  and  Fertilization  (chap. 
xix),  we  need  not  consider  this  matter  in  detail. 

A  mere  negative  finding  in  this  type  of  experiment 
is,  however,  inconclusive.     It  is  conceivable,  as  Loeb 


134  PROBLEMS  OF  FERTILIZATION 

has  pointed  out,  that  the  postulated  fertilizing  sub- 
stance of  the  spermatozoon  requires  the  motive  power 
of  the  spermatozoon  to  make  it  effective  against  the 
egg,  which  is  the  only  indicator,  and  that  it  will  not 
act  in  solution;  it  is  also  possible  that  it  is  labile  in 
solution,  or  that  methods  employed  in  the  endeavor  to 
isolate  it  have  been  too  brutal.  We  have  seen  (pp.  1 12  ff .) 
that  spermatozoa  carry  an  agglutinable  substance  which 
is  lost  after  reaction  with  the  agglutinating  substance  of 
the  egg;  the  presence  of  such  a  substance  could  not  be 
demonstrated  by  mere  isolation  methods. 

The  writer  (191 5)  has  employed  another  method  for 
demonstrating  the  existence  of  such  a  substance.  He 
showed  that  the  rate  of  loss  of  fertilizing  power  of 
sperm  suspensions  of  ^r^aaa  is  inversely  proportional  to 
their  concentration  and  that  in  great  dilutions  the  loss 
of  fertilizing  power  is  many  times  more  rapid  than  the 
loss  of  motility.  This  was  ascribed  to  loss  of  a  specific 
substance  by  the  spermatozoon,  for  the  other  factors 
for  successful  insemination  were  maintained  constant. 
Other  observers  (e.g.,  Schiicking,  1903,  and  Glaser, 
1 9 14)  have  noted  that  several  spermatozoa  appear  to 
be  needed  for  fertilization  of  a  single  ovum,  because 
fertilization  usually  failed  under  the  conditions  of  their 
experiments  unless  a  number  of  spermatozoa  were  pres- 
ent simultaneously  in  the  jelly  around  the  egg.  Glaser 
postulated  a  mass  effect  of  spermatozoa  in  fertilization 
as  well  as  an  individual  effect  to  explain  this.  The 
writer  could,  however,  demonstrate  that  mass  effect  is 
not  necessary  if  the  sperm  suspensions  are  sufficiently 
fresh,  and  that  the  appearance  observed  by  Schiicking  and 
Glaser  is  found  only  with  suspensions  not  perfectly  fresh. 


THE  PHYSIOLOGY  OF  FERTILIZATION  135 

The  fertilizing  power  of  sperm  suspensions  may  be 
expressed  in  curves  whose  ordinates  are  percentages  of 
fertilization,  and  the  abscissae  a  geometrical  series  of 
dilutions  of  i  per  cent  sperm  in  powers  of  2.  This 
method  was  adopted  for  the  abscissae  because  of  the 
method  of  successive  half-dilutions  used  in  many  experi- 
ments, and  because  the  enormous  range  of  fertilizing 
power  made  it  impossible  to  compare  results  on  one 


lOo 
90 
80 
70 
60 
SO 
40 
30 
20 
10 
o  L 


'   '   ^   ■*   S   6   7   8   9  10  fr  12  13  14  IS  16  17  18  15  20  21  22 


23  24  25  26  27 


Fig.  16. — Curve  of  fertilizing  power  of  perfectly  fresh  sperm 
suspensions  oiArbacia.  The  ordinates  give  percentages  of  eggs  fertilized. 
The  abscissae  represent  dilutions  of  the  sperm  in  powers  of  2,  viz.: 
1  =  1;   2  =  i^;  3  =  i3,  etc. 

scale  with  an  arithmetical  progression.  When  it  is 
realized  that  fertilizing  power  may  extend  to  1/90,000,- 
000  of  I  per  cent  the  necessity  of  the  geometrical  series 
in  the  abscissae  will  bedome  apparent. 

Figure  16  is  prepared  from  data  of  experiments  calcu- 
lated to  bring  eggs  and  sperm  together  in  the  freshest 
possible  condition  of  the  sperm.  In  general  measured 
quantities  of  washed  eggs  were  put  in  measured  amounts 
of  sea-water,  and  measured  quantities  of  definitely  cali- 
brated sperm  suspensions  added  and  stirred  in  as  uni- 
formly as  possible.     A  control  of  unfertilized  eggs  in 


136  PROBLEMS  OF  FERTILIZATION 

sea-water  was  always  kept  to  guard  against  chance 
fertilizations.  To  illustrate:  the  last  four  detennina- 
tions  of  the  curve  were  made  as  follows :  In  four  crystal- 
lization dishes  were  placed  1,000  c.c.  sea-water  (A), 
3,000  c.c.  sea-water  (B),  1,000  c.c.  sea- water  (C),  3,000 
c.c.  sea-water  (D).  To  each  was  added  2  c.c.  of  a 
washed  egg  suspension.  The  sperm  was  then  prepared 
as  follows:  (i)  one  drop  dry  spenii  to  3.3  c.c.  sea- 
water  at  9:43  A.M.  =  1  per  cent;  (2)  i  c.c.  sperm  i 
to  99  c.c.  sea-water  at  9:43^  a.m.  =  1/100  per  cent; 
(3)  I  c.c.  sperm  i  to  999  c.c.  sea-water  at  9:45^  a.m.= 
1/1,000  per  cent.  To  A  was  added  i  drop  sperm  2 
(i/ioo  per  cent)  at  9:43!;  to  B  one  drop  sperm  2 
(i/ioo  per  cent)  at  9:44;  to  C  one  drop  sperm  3  (1/1,000 
per  cent)  at  9:45!;  to  D  one  drop  sperm  3  (1/1,000  per 
cent)  at  9:45!.  An  assistant  stirred  in  the  sperm  thor- 
oughly as  added.  The  sperm  concentration  in  A  was 
therefore  1/100X1/30X1/1,000=1/3,000,000  per  cent; 
in  B  it  was  1/9,000,000  per  cent;  in  C,  1/30,000,000 
per  cent;  in  D,  1/90,000,000  per  cent;  1/3,000,000 
per  cent  falls  between  21  and  22  on  the  scale,  and  the 
others  as  shown.  The  exact  times  of  mixing  the  sperm 
are  given  because,  as  will  appear  beyond,  time  is  an 
extremely  important  factor  with  reference  to  fertilizing 
power. 

To  appreciate  the  extent  of  the  greatest  dilution  it 
may  be  said  that  beyond  a  dilution  of  1/10,000  per  cent 
(between  13  and  14  on  the  scale)  one  can  rarely  find  a 
single  spermatozoon  in  the  jelly  of  the  fertilized  eggs. 
At  about  1/2,000  per  cent  (11  on  the  scale)  the  sperm 
suspension  does  not  even  appear  opalescent.  We  may 
therefore  feel  reasonably  sure  that  beyond  about  14  or 


THE  PHYSIOLOGY  OF  FERTILIZATION 


137 


8    .9      10     II 


15  on  the  scale  no  egg  will  receive  more  than  one  sper- 
matozoon. 

In  further  elucidation  of  the  curve  (Fig.  16)  I  may 
say  that  the  critical  (steep)  part  was  covered  by  several 
determinations  for  each  point.  Thus  there  are  five 
determinations  averaged  for  the  positions  between  13 
and  15,  seven  between  15  and  18,  five  between  18  and 
20,  and  six  between  20  and  21.  The  determinations 
beyond  2 1  are  single  deter- 
minations. For  the  first 
part  of  the  curve  up  to 
13  there  are  numerous 
determinations.  There 
are  great  variations  in 
the  single  determina- 
tions compared  with  one 
another;  these  averages 
must  therefore  be  re- 
garded only  as  approxi- 
mate values.  With  a 
sufficiently  large  number 


90 
80 
70 
60 
50 
40 
30 


______ 


Fig.  17. — Curve  of  fertilizing  power 
of  sperm  suspensions  of  .4  r6ac/'a  about 
20  minutes  old.  Ordinates  and  ab- 
scissae as  in  Fig,  16. 


of  determinations  the  irregularities  between  15  and  17 
and  between  19  and  22  would  no  doubt  disappear.  But 
it  is  improbable  that  the  general  form  of  the  curve 
would  undergo  any  essential  change  even  with  a  much 
more  extensive  series  of  determinations. 

If  now  we  compare  this  curve  of  fertihzation  with 
perfectly  fresh  sperm  with  sperm  suspensions  about 
twenty  minutes  old  we  get  the  result  shown  in  Fig.  17. 
It  will  be  seen  that  fertilizing  power  ceases  at  |"*  after 
thirty  minutes.  If  now  one  determines  by  compari- 
son  the  rate  of  complete  loss  of  fertilizing  power  of 


138  PROBLEMS  OF  FERTILIZATION 

sperm  suspensions  of  different  concentrations  it  is 
found  that  sperm  suspensions  of  1/240,000  per  cent 
decline  to  zero  in  their  fertilizing  power  in  about  six 
minutes,  those  of  1/30,000  per  cent  in  about  sixteen 
minutes,  those  of  1/300  per  cent  not  until  after  more 
than  two  hours,  while  i  per  cent  sperm  may  maintain 
fertilizing  power  for  two  or  more  days. 

At  the  time  when  sperm  suspensions  are  losing  their 
fertilizing  power  one  can  observe  the  phenomenon 
described  by  Schlicking  and  Glaser  at  the  proper 
concentration  of  sperm.  Thus  in  one  of  the  writer's 
experiments  eggs  were  added  to  a  1/128  per  cent  sperm 
suspension  that  was  on  the  point  of  complete  loss  of 
fertilizing  power.  In  ten  eggs  selected  at  random  an 
average  of  nine  spermatozoa  was  counted  in  the  jelly 
in  an  optical  section.  The  spermatozoa  were  still 
active,  but  the  eggs  did  not  fertilize. 

Gemmill  (1900)  observed  a  relation  between  dura- 
tion of  fertilizing  power  and  concentration  of  sperm  in 
the  sea  urchin  and  concluded  that  the  more  rapid 
exhaustion  of  spermatozoa  in  dilute  suspensions  is  due 
to  dilution  of  a  hypothetical  nutritive  medium  which 
keeps  the  spermatozoa  of  concentrated  suspensions  in 
a  vigorous  condition.  This  explanation  is  not  only 
purely  hypothetical  but  comes  back  to  the  principle 
of  loss  of  motility,  which,  as  we  have  seen,  cannot  apply 
in  this  case. 

The  writer  therefore  holds  that  spermatozoa  tend 
to  lose  their  fertilizing  substance  in  proportion  to  dilu- 
tion, so  that  they  may  thus  become  ineffective,  whatever 
their  motility.  For  reasons  discussed  later  it  seems 
probable  that  this  fertilizing  substance  is  identical  with 


THE  PHYSIOLOGY  OF  FERTHJZATION  139 

the  agglutinablc  substance  of  the  spermatozoon,  which 
is  apparently  lost  by  staling,  as  we  have  previously 
seen. 

The  main  principle  of  this  discussion,  viz.,  that 
spermatozoa  may  lose  their  fertilizing  power  for  other 
causes  than  loss  of  motility,  or  that  motility  alone  is 
not  an  adequate  criterion  of  fertilizing  power  of  sper- 
matozoa, has  obvious  important  bearings.  The  mere 
fact  that  spermatozoa  may  retain  their  motility  for 
three  weeks  or  more  in  the  human  genital  tract  (Wal- 
deyer,  1906)  by  no  means  proves  that  they  retain  their 
fertiHzing  power  during  all  this  time,  although  this  has 
been  almost  universally  assumed.  In  an  excellent  paper 
published  after  his  death.  Mall  (1918)  points  out  the 
many  contradictions  and  unnecessary  assumptions  that 
this  belief  entails  with  reference  to  the  facts  of  human 
conception,  and  he  concludes  that  it  is  probable  that 
spermatozoa  have  lost  their  fertilizing  power  by  the 
time  they  have  passed  the  tube.  Bryce  and  Teacher 
(1908)  and  Triepel  (1914-15)  also  conclude  that  ferti- 
lization must  occur  within  forty-eight  hours  after 
copulation. 

2.  The  ovum. — Ova  attain  a  fertilizable  condition 
rather  suddenly,  as  a  rule  at  the  very  end  of  the  period 
of  growth  or  at  the  beginning  of  the  maturation  period. 
The  egg  of  Nereis  is  fertilizable  before  the  rupture 
of  the  germinal  vesicle,  but  the  ova  of  sea  urchins, 
starfish,  Dentalium  (mollusk),  and  Nemerteans  are 
not  fertilizable  until  the  germinal  vesicle  has  begun 
to  break  down  (see  section  on  merogony).  Ova  may, 
however,  be  penetrated  by  spermatozoa  at  an  earlier 
period  but  without  any  fertilization  reaction  occurring. 


I40  PROBLEMS  OF  FERTILIZATION 

O.  and  R.  Hertwig  (1887)  were  the  first  to  observe  this: 
under  the  influence  of  chloral  hydrate  spermatozoa  pen- 
etrate unripe  sea  urchin  eggs  in  large  numbers,  but  the 
eggs  remain  unchanged  and  the  sperm  heads  undergo  no 
change  within  the  egg.  Under  normal  conditions  sper- 
matozoa do  not  usually  penetrate  into  such  unripe  eggs. 
The  writer  has  observed  penetration  of  unripe  ovocytes 
of  Chaetopterus  without  any  subsequent  reaction  of  egg 
or  sperm.  Wilson  (1903)  has  observed  for  Cerebratuliis, 
and  Delage  (1901a)  for  the  starfish,  that  enucleated 
portions  of  ova,  full  grown  but  with  intact  germinal 
vesicle,  will  not  give  any  fertilization  reaction,  but  as 
soon  as  the  germinal  vesicle  has  broken  down  similar 
pieces  readily  fertilize. 

Fertilization  capacity  thus  arises  suddenly  in  ova. 
The  most  natural  working  hypothesis  is  that  this  is 
due  to  formation  of  a  definite  substance  essential  for 
fertilization.  This  is  a  conception  that  we  shall  exam- 
ine more  fully  later  on.  For  purposes  of  reference  we 
shall  call  this  (for  the  present)  h^^^othetical  substance 
fertilizin. 

The  fertilizable  condition  of  the  ovum  is  not  of 
indefinite  duration.  Indeed  in  many  cases  its  duration 
is  exceedingly  brief.  The  most  remarkable  case  of  this 
kind  is  found  in  the  annelid  Platynereis  megalops, 
which  has  been  very  beautifully  analyzed  by  E.  E. 
Just  (191 5).  Fertihzation  is  normally  internal  in  this 
animal  and  the  eggs  are  laid  as  soon  as  fertilized. 
Artificial  insemination  may  be  successfully  performed 
by  mixing  the  ova  and  spermatozoa  dry,  i.e.,  without 
any  contact  with  sea- water  until  after  insemination; 
but  sea-water  may  be  added  5  seconds  later.     If,  how- 


THE  PHYSIOLOGY  OF  FERTILIZATION  141 

ever,  the  eggs  are  placed  in  sea-water  for  even  a  few 
seconds  before  insemination  they  cannot  be  normally 
fertilized  even  if  the  sea-water  be  filtered  off.  The 
effect  may  be  graded  by  using  minimal  quantities  of 
sea-water.  But  the  sperm  can  remain  in  sea-water  for 
some  time,  and  after  filtering  off  the  sea-water  they 
will  normally  fertilize  dry  eggs.  Washed  eggs  will  not 
fertilize,  but  washed  sperm  will. 

The  time  factor  involved  in  these  experiments  is 
far  too  short  to  give  any  support  to  the  assumption 
that  sea-water  ''injures"  the  eggs  in  other  ways;  it 
is  also  impossible  to  postulate  a  membrane  effect  of 
the  sea-water  inhibiting  entrance  of  the  spermatozoon, 
because  the  effect  can  be  graded  readily  so  as  to  permit 
penetration,  but  in  this  case  cleavage  does  not  occur. 
The  results  permit  of  only  one  conclusion,  viz.:  that 
unfertilized  eggs  lose  in  sea-water  a  substance  necessary 
for  fertilization.  Just  was  also  able  to  demonstrate  a 
substance  in  the  sea-water  used  for  washing  the  eggs 
that  has  an  agglutinating  effect  on  both  Platy nereis  and 
Nereis  sperm. 

The  case  of  Platy  nereis  is  unusual  with  reference  to 
the  very  brief  duration  of  the  fertilizable  condition  of 
the  ovum  in  the  medium  for  development.  But  there 
are  many  other  forms  in  which  the  fertilizable  period 
is  very  brief.  For  instance,  Reighard  (1893)  states 
that  it  is  not  possible  to  fertilize  the  eggs  of  wall-eyed 
pike  that  have  lain  in  water,  and  he  gives  the  follow- 
ing table  (p.  142)  in  order  to  substantiate  the  state- 
ment. ''Five  lots  of  twenty-five  eggs  each  were  prepared 
for  fertilization  by  placing  them  in  watch  glasses  in  the 
usual  way  and  to  each  lot,  after  it  had  been  a  certain 


142  PROBLEMS  OF  FERTILIZATION 

time  in  water,  was  was  added  freshly  prepared  milt  as 
follows : 

Percentage  of 
Milt  Added  Fertilized  Eggs, 

after  i.e.,  Segmented 

Lot  I 2  min.  40 

Lot  2 4  min.  17 

Lot  3 6  min.  10 

Lot  4 8  min.  5 

Lot  5 10  min.  o  " 

Even  two  minutes  in  water  has  great  detrimental  effect, 
and  at  ten  minutes  all  eggs  have  lost  capacity  for  ferti- 
lization. Similarly  the  egg  of  the  frog  is  incapable  of 
being  fertilized  after  lying  in  water  a  short  time  (Spal- 
lanzani  and  others). 

We  can  explain  such  cases,  as  already  suggested, 
by  the  loss  of  the  hypothetical  fertihzin.  Other  eggs 
are  apparently  better  protected  with  reference  to  their 
fertilizin  content.  Starfish  eggs  rapidly  lose  capacity 
for  fertihzation  after  separation  of  the  first  polar  body. 
The  egg  of  the  sea  urchin  will  bear  repeated  washings 
in  sea-water  without  loss  of  fertilization  capacity;  but 
after  a  certain  number  of  washings  before  insemination 
the  developmental  energy  of  the  fertilized  eggs  becomes 
progressively  reduced.  The  writer  regards  the  gelat- 
inous covering  of  these  eggs  as  a  protection  against 
loss  of  fertilizin,  and  has  shown  (chap,  vii)  that  after 
complete  rerhoval  of  jelly  sea  urchin  eggs  are  much 
less  resistant  against  loss  of  fertilizing  capacity  by 
repeated  washings.  Loeb  has  suggested  that  loss  of 
vitahty  would  account  for  the  result  (191 5,  p.  283), 
but,  apart  from  the  vagueness  of  the  suggestion,  there 
are  other  reasons  for  assigning  a  more  specific  cause, 
which  will  be  discussed  later. 


THE  PHYSIOLOGY  OF  FERTILIZATION  143 

Finally  all  eggs  completely  lose  fertilization  capacity 
after  fertilization,  a  problem  that  we  shall  discuss  in 
chapter  vii. 

We  have  thus  seen  reason  to  believe  that  the  ferti- 
lizable  condition  of  gametes  is  associated  with  the  pres- 
ence of  a  definite  substance  in  each.  The  evidence  is 
perhaps  more  conclusive  in  the  case  of  the  ovum  than 
in  the  case  of  the  spermatozoon.  But  we  are  justified 
in  using  this  conception  as  a  working  hypothesis,  and 
it  will  be  found  very  useful  in  subsequent  analysis. 

The  capacity  for  parthenogenesis  also  exhibits  par- 
allel relations  to  fertilization  capacity.  Delage  (1901a) 
was  the  first  to  notice  this;  in  his  experiments  on  cyto- 
plasmic maturation  in  the  egg  of  the  starfish  he  notes 
that  capacity  for  fertilization  arises  first  with  the  break- 
ing down  of  the  germinal  vesicle;  this  condition  lasts 
until  the  appearance  of  the  first  polar  body,  but  when  the 
second  polar  body  is  about  to  appear  fragments  are  less 
fertilizable,''even  if  they  do  not  become  completely  resist- 
ant to  merogonic  fertilization."  He  finds  a  similar  criti- 
cal period  for  parthenogenesis ;  shortly  after  the  germinal 
vesicle  breaks  down,  parthenogenic  agents  are  peculiarly 
efficacious;  this  gradually  disappears  before  the  second 
polar  body  is  formed.  ''There  is  a  single  point  in  the 
physiological  curve  of  the  egg  when  the  least  disturbing 
action  may  cause  it  to  turn  toward  parthenogenesis." 

R.  S.  Lillie  (191 5)  has  made  a  more  detailed  exam- 
ination of  this  point  with  reference  to  heat  partheno- 
genesis in  the  eggs  of  the  starfish.  A  short  exposure  of 
the  eggs  to  temperatures  between  32°  and  ^S°  C.  will 
cause  the  development  of  almost  every  egg  to  a  free- 
swimming  larval  stage;    for  each  temperature  there  is 


144  PROBLEMS  OF  FERTILIZATION 

a  well-defined  optimum  exposure,  and  the  temperature 
coefiicient  is  over  loo.  ''The  responsiveness  of  the  eggs 
to  this  form  of  treatment  was  found  to  depend  on  the 
stage  of  maturation;  warming  before  the  dissolution 
of  the  germinal  vesicle  had  begun  was  ineffective,  and 
in  fact  inhibited  maturation  entirely;  the  most  favor- 
able period  lay  between  the  breakdown  of  the  germinal 
vesicle  and  the  separation  of  the  first  polar  body;  after 
both  polar  bodies  had  separated  development  was 
imperfect  and  never  proceeded  far, — even  membrane 
formation  then  failed  in  many  eggs." 

Thus  while  the  eggs  of  the  starfish  are  still  in  a 
condition  of  unimpaired  vitality,  as  is  shown  by  contin- 
uation of  the  maturation  divisions,  they  lose  capacity 
both  for  fertilization  and  parthenogenesis. 

The  inference  that  the  failure  to  respond  to  parthe- 
nogenetic  agents  is  due  to  loss  of  some  substance  in 
sea- water  was  strongly  supported  by  Just  (igi^b)  in 
a  study  of  heat  parthenogenesis  in  Nereis.  He  found 
that  eggs  of  Nereis  which  were  first  washed  in  sea-water 
could  not  be  induced  to  develop  by  exposure  to  warmed 
sea-water,  or  only  an  exceedingly  small  percentage  are 
affected;  if,  however,  the  eggs  are  exposed,  without 
previous  contact  with  sea-water,  to  a  favorable  temper- 
ature for  an  optimum  time  in  a  small  quantity  of  sea- 
water,  all  of  them  may  segment,  and  as  many  as  20  per 
cent  may  develop  into  trochophores.  Just  found  that 
capacity  for  heat  parthenogenesis  is  lost  much  more 
rapidly  in  sea- water  than  capacity  for  fertilization,  but 
the  latter  is  also  affected,  though  at  a  lesser  rate.  These 
capacities  he  found  to  run  parallel  to  loss  of  sperm- 
agglutinating  substance  by  the  eggs. 


THE  PHYSIOLOGY  OF  FERTILIZATION  145 

III.    PHYSIOLOGICAL   INDICIA    OF    THE    FERTILIZATION 

REACTION 

We  have  already  considered  the  morphological  signs 
of  fertilization  but  may  again  summarize  them:  (i) 
There  are  invariably  certain  cortical  morphological 
changes,  such  as  formation  of  a  fertilization  membrane 
(echinoderms  and  nematodes),  formation  of  a  perivi- 
telline  space  (most  animals),  and  secretion  of  jelly 
{Nereis,  Platynereis) .  (2)  Maturation  is  set  in  process 
or  resumed,  provided  it  is  not  complete  before  fertili- 
zation. (3)  In  the  egg  a  sperm  aster  is  usually  formed, 
the  sperm  nucleus  enlarges  and  metamorphoses,  and  the 
germ  nuclei  unite.  What  are  the  immediate  physio- 
logical consequences  or  indicia  ? 

I.  Changes  in  rate  of  oxidation. ~~V^2irh\xrg  (1908-14) 
determined  that  the  rate  of  consumption  of  oxygen  by 
fertilized  eggs  of  the  sea  urchin  is  six  to  seven  times 
that  of  unfertilized  eggs,  and  that  the  rate  of  oxygen 
consumption    increases    progressively    for    some    time. 
Loeb  and  Wastenys   (191 2-13)   found  the  increase  in 
fertilized   eggs  of  Strongylocentrotus  purpiiratus   to   be 
four  or  five  to  one  as  compared  with  unfertilized  eggs. 
But  they  did  not  find  any  significant  change  in  oxygen 
consumption    in    the    starfish    egg    after    fertilization 
(191 2).     These  authors  also  determined  that  membrane 
formation  by  artificial  means  causes  a  comparable  in- 
crease^ in    the    rate    of    oxidation.     The    materials    of 
comminuted  unfertilized  eggs  of  sea  urchins  (mechanical 
[Warburg,  1914],  or  by  cytolysis  [Loeb  and  Wastenys, 
1913])  also  show  an  equal  or  greater  increase  in  oxygen 
consumption  as  compared  with  an  equal   amount  of 
intact  unfertilized    eggs.      It   would   therefore   appear 


146  PROBLEMS  OF  FERTILIZATION 

that  the  material  of  unfertilized  eggs  is  in  a  highly  oxi- 
dizable  condition.  Comminuted  material  of  fertilized 
eggs  consumes  no  more  oxygen  than  intact  fertilized 
eggs. 

The  inference  has  been  drawn  from  these  oxidation 
studies  that  the  same  metabolic  activities  present  before 
fertilization  are  accelerated,  and  that  this  is  the  real 
essence  of  activation.  But  the  fact  that  in  the  star- 
fish there  is  no  measurable  increase  in  oxidation  after 
fertilization,  although  we  have  the  same  phenomenon 
of  initiation  of  development  (activation),  sets  such  a 
conclusion  in  a  rather  doubtful  light.  It  is  probable 
that  in  the  case  of  the  starfish  there  is  an  increase  in 
the  rate  of  oxidation  during  maturation,  thus  before  and 
independent  of  fertilization.  The  connection  between 
fertilization  and  increase  of  oxidation  may  thus  be 
incidental.  If  we  realize  that  the  egg  is  a  highly 
complex  system,  and  that  the  rate  of  oxidation  as 
measured  is  a  gross  result  without  any  distinction  of 
kind  or  location,  the  obvious  alternative  stands  out 
that  we  may  be  measuring  quite  different  metabolic 
activities  before  and  after  fertilization.  If  this  were 
the  case,  it  is  obvious  that  the  net  result  in  terms  of 
oxygen  consumption  might  not  change  or  might  show 
either  a  gain  or  a  loss,  depending  on  the  nature  of  the 
system.  There  may  be  a  retardation  of  some  processes 
and  an  acceleration  of  others,  giving  a  different  net 
result  in  the  sea  urchin  and  starfish,  for  instance. 
The  mere  determination  of  gain  in  oxygen  consump- 
tion, even  if  it  were  a  universal  fact,  is  not  adapted 
to  carry  us  very  far  in  the  problem  of  fertilization 
without  a  more  minute  analysis. 


THE  PHYSIOLOGY  OF  FERTILIZATION  147 

2.  Changes  in  permeability. — The  increase  in  oxygen 
consumption  of  fertilized  eggs  of  the  sea  urchin  indi- 
cates a  readier  access  of  oxygen  to  the  material  of  the 
Qgg,  and  not  a  change  in  the  oxidizable  character  of  the 
material,  because  the  comminuted  material  of  the  unferti- 
lized egg  also  consumes  more  oxygen  than  the  intact  Qgg. 
The  egg  membrane  must  therefore  be  more  permeable  to 
oxygen  after  fertilization  than  before  in  those  cases  in 
which  increase  of  oxidation  after  fertilization  occurs. 
There  are  several  other  evidences  of  an  increase  in  perme- 
ability of  the  egg  membrane  as  a  result  of  fertilization. 

Among  these  is  the  escape  of  substances  from  the 
egg  which  previously  escaped  more  slowly  or  not  at 
all.  Carbon  dioxide  is  an  example  of  the  first  kind; 
there  is  a  sudden  increase  of  CO2  production  at  the  time 
of  fertilization  in  the  sea  urchin  corresponding  to  in- 
creased oxygen  consumption  (Lyon).  As  an  example 
of  the  second  kind  the  escape  of  pigment  from  the  eggs 
of  Arbacia  at  the  time  of  fertilization  may  be  noted; 
Lyon  and  Shackell  (1910)  have  described  this  phenome- 
non, which  has  also  been  observed  by  other  investi- 
gators. The  extrusion  of  jelly  from  the  egg  of  Nereis 
previously  described  (p.  53)  is  another  striking  instance; 
the  egg  of  Ascaris  megalocephala  similarly  excretes  a 
considerable  amount  of  material  at  fertilization  which 
forms  the  thick  resistant  egg  membrane  of  this  form. 
According  to  Reighard  (1893)  the  egg  of  the  wall-eyed 
pike  similarly  excretes  a  substance  from  its  cortical 
layer  at  the  time  of  fertilization,  and  the  same  is  prob- 
ably true  of  teleosts  in  general. 

Diminution  in  the  size  of  the  egg  following  fertili- 
zation due  to  loss  of  materials  has  also  been  carefully 


1 48  PROBLEMS  OF  FERTILIZATION 

measured  by  Glaser  (1914);  individual  eggs  of  Arhacia 
were  measured  before  and  after  fertilization,  and  in 
nearly  all  cases  a  measurable  decrease  in  diameter  was 
recorded,  in  some  cases  as  much  as  10  per  cent.  In 
the  starfish  the  decrease  was  found  to  be  still  greater. 
The  contrary  results  of  McClendon  (19 10)  on  Arhacia 
may  be  interpreted  as  due  to  the  great  individual  varia- 
tions in  size  of  the  eggs,  which  were  measured  in  a 
statistical  and  not  in  an  individual  way. 

Loeb  (1908)  believes  that  to  explain  the  tension  of 
the  fertilization  membrane  (in  Strongylocentrotus)  it  is 
necessary  to  assume  some  slight  loss  of  colloids  from  the 
egg  at  fertilization,  but  he  was  unable  to  detect  any 
measurable  difference  in  the  diameter  of  fertilized  and 
unfertilized  eggs.  Okkelberg  (19 14)  has  determined  by 
careful  measurements  of  individual  eggs  that  fertiliza- 
tion produces  an  average  decrease  of  13.48  per  cent  in 
the  volume  of  the  eggs  of  the  brook  lamprey.  He 
thinks  that  the  substances  lost  include  some  colloid 
material. 

The  increase  in  permeability  resulting  from  fertiliza- 
tion has  also  been  tested  in  other  ways.  McClendon 
(1910)  demonstrated  an  increase  of  electrical  conductiv- 
ity as  a  result  of  fertilization  or  action  of  partheno- 
genetic  agents  in  the  egg  of  Toxopneustes;  he  determined 
the  resistance  (reciprocal  of  conductivity)  of  unferti- 
lized eggs  to  be  595  ohms  and  that  of  the  same  eggs 
after  fertilization  to  be  455  ohms;  from  this  he  argues 
that  the  egg  becomes  more  permeable  to  ions,  on  which 
the  conductivity  depends,  at  the  beginning  of  devel- 
opment. Gray  (1913)  made  similar  determinations. 
Lyon  and  Shackell  (1910)  have  also  shown  that  eggs 


THE  PHYSIOLOGY  OF  FERTILIZATION  149 

become  more  permeable  to  certain  intra-vitam  dyes 
immediately  after  fertilization.  Harvey  (19 10)  con- 
firmed this  and  showed  also  a  temporary  increase  in  the 
permeability  toward  alkahes.  Lyon  (1909)  shows  that 
about  double  the  amount  of  oxygen  is  liberated  from 
H2O2  by  fertilized  eggs  of  sea  urchins  as  compared  with 
equal  amounts  of  unfertilized  eggs,  beginning  about 
three  minutes  after  insemination.  This  may  be  ex- 
plained by  increased  permeability  by  which  peroxide 
and  catalase  come  more  easily  together. 

R.  S.  Lillie  (1916-18)  has  shown  that  after  fertili- 
zation Arhacia  eggs  take  up  water  by  osmosis  several 
times  more  rapidly  than  before  fertilization;  if  a  mix- 
ture of  fertilized  and  unfertilized  eggs  be  placed  in 
hypotonic  sea-water  (forty  parts  sea-water  plus  sixty  of 
tap  water)  it  is  possible  within  two  or  three  minutes  to 
distinguish  the  fertilized  eggs  by  their  greater  diameter. 
The  volume  of  water  entering  the  fertilized  egg  in  three 
minutes  was  found  to  be  iiXio^/x^  and  the  unfertiHzed 
egg,  3  .6Xio^ju^.  Osmotic  equilibrium  is  reached  within 
about  eight  minutes  in  the  case  of  the  fertilized  egg; 
in  the  case  of  the  unfertilized  egg  the  entrance  of  water 
is  more  gradual,  and  many  of  the  eggs  break  down 
before  osmotic  equilibrium  is  reached.  Figure  18  fur- 
nishes a  comparison  of  the  rate  of  entrance  of  water. 

The  rate  of  entrance  of  water  is  essentially  constant 
during  the  period  of  exposure  in  the  case  of  both  ferti- 
lized and  unfertilized  eggs  relative  to  the  existing  gra- 
dient of  osmotic  pressure,  which  demonstrates  that  the 
differences  between  the  two  sorts  is  due  to  a  difference 
in  the  resistance  of  the  membrane  to  the  passage  of 
water.     The  amount  of  water  which  enters  or  leaves 


i5<^ 


PROBLEMS  OF  FERTILIZATION 


the  cell  is  finally  the  same  in  both  fertilized  and  unfer- 
tilized eggs,  thus  again  demonstrating  a  membrane 
effect.  Eggs  which  have  been  treated  with  partheno- 
genetic  agents  show  an  increase  of  permeability  compa- 
rable to  that  of  fertilized  eggs,  but  of  a  more  fluctuating 
character. 


Fig.  1 8. — Imbibition  of  water  by  Arhacia  eggs  in  diluted  sea- water 
(60  volumes  tap  water  plus  40  volumes  sea-water).  The  curves  are 
measurements  of  diameters  at  different  intervals  after  placing  in  diluted 
sea-water.  Ordinates  are  diameters  in  micra,  abscissae  minutes  after 
placing  in  the  hypotonic  medium.  A,  fertilized  eggs;  B,  eggs  with 
artificial  membranes;   C,  unfertilized  eggs  (after  R.  S.  Lillie). 


In  the  reverse  experiment,  of  subjecting  fertilized 
and  unfertilized  eggs  to  the  action  of  hypertonic  sea- 
water,  R.  S.  Lillie  (19 18)  has  shown  that  fertilized 
eggs  lose  water  much  more  rapidly,  as  is  to  be  expected; 
and  the  two  kinds  of  eggs  can  thus  be  separated  by 
gravity  in  tall  tubes  of  hypertonic  sea-water. 


THE  PHYSIOLOGY  OF  FERTILIZATION  151 

The  condition  of  greater  permeability  to  water  does 
not  arise  all  at  once,  but  gradually  during  the  first 
fifteen  minutes  after  insemination;  this  is  a  matter  of 
great  significance  in  considering  the  relation  of  change 
of  permeability  to  the  fertilization  reactions. 

The  behavior  of  the  fertihzed  and  unfertilized  eggs 
of  Echinarachnins  to  hypotonic  and  h^-pertonic  sea- 
water  is  similar  to  that  of  Arhacia;  but  the  starfish 
{Asterias)  shows  an  interesting  contrast  to  both,  as  there 
is  httle  difference  between  fertilized  and  unfertilized 
eggs  with  reference  to  exosmosis  and  endosmosis  (R.  S. 
LilHe,  19 18).  A  comparable  contrast  with  reference  to 
oxygen  consumption  in  the  sea  urchin  and  starfish  eggs 
respectively  has  already  been  noted  (p.  145). 

R.  S.  Lillie  (191 7)  distinguishes  two  possibilities 
with  reference  to  the  relation  which  changes  of  per- 
meabihty  may  bear  to  the  activation  process. 

The  first  possibility  is  that,  after  fertilization  and  as  one  of 
the  secondary  consequences  of  this  process,  the  general  condi- 
tions of  permeability  in  the  egg  are  permanently  modified,  and 
that  the  protoplasmic  surface-layer  or  plasma-membrane  from 
that  time  on  remains  more  permeable  and  more  subject  to  changes 
of  permeabiHty  than  before;  there  is,  in  fact,  considerable  evi- 
dence that  this  is  the  case.  The  second  possibility,  which  is 
the  one  suggested  by  the  resemblances  between  the  conditions 
of  activation  of  the  resting  egg  and  those  of  stimulation  in 
general,  is  that  the  primary  event  in  the  activation  process,  as 
well  as  in  the  stimulation  process,  is' a  temporar>'  increase  of 
permeabihty;  upon  this  initial  change  follow  the  other  changes 
expressive  of  the  general  response  or  activation  of  the  egg-cell. 
A  temporary  or  initiatory  increase  of  permeability  is  thus  to  be 
distinguished  from  a  permanent  alteration  in  the  general  proper- 
ties of  the  plasma-membrane  involving  increased  permeability. 
There  is  good  reason  to  believe  that  both  of  these  processes  are 
concerned  in  the  activation  of  the  resting  egg. 


152  PROBLEMS  OF  FERTILIZATION 

Summarizing  this  section  we  have  the  following 
evidences  of  increase  of  permeability  of  the  plasma 
membrane  at  the  time  of  fertilization:  increase  of 
oxygen  consumption,  escape  of  substances  from  the  egg 
at  the  time  of  fertilization  (increased  CO^,  pigment, 
jelly  in  Nereis,  etc.),  decrease  in  volume  of  the  egg, 
increase  of  electrical  conductivity,  increased  permeabil- 
ity to  certain  intra-vitam  stains,  greater  catalytic 
power  of  fertilized  eggs,  increased  permeability  to  alka- 
lies, increase  in  rate  of  osmotic  change  with  reference 
to  water. 

There  can  be  no  doubt  that  the  permeability  of  the 
egg  increases  at  the  time  of  fertilization  in  some  cases 
at  least.  On  the  other  hand  it  is  certain  that  this 
change  is  less  in  some  eggs  than  in  others.  No  doubt 
the  great  differences  in  vitality  of  unfertilized  eggs  of 
different  species  are  dependent,  in  part  at  least,  on 
such  variations.  Later  on  we  shall  discuss  the  role  of 
permeability  changes  in  the  complex  of  fertilization 
processes. 

3.  Changes  in  physical  state. —Mdiny  observers  have 
noted  that  changes  in  the  physical  state  of  the  proto- 
plasm of  the  egg  accompany  fertilization.  Thus  the 
eggs  of  Ascaris  megalocephala ,  which  have  an  irregular 
outhne  prior  to  fertilization,  become  definitely  spherical 
soon  after  the  entrance  of  the  spermatozoon  as  the 
fertilization  membrane  forms;  on  the  other  hand  the 
eggs  of  Nereis,  which  have  a  very  regular  contour  before 
fertilization,  become  decidedly  irregular  in  outline  fifteen 
to  twenty  minutes  after  insemination,  and  gradually 
resume  the  spherical  state  before  the  first  polar  body  is 
formed.     A  decided  difference   in  the  behavior  of  the 


THE  PHYSIOLOGY  OF  FERTILIZATION  153 

cytoplasm  to  mechanical  shocks  (e.g.,  shaking)  has  been 
similarly  noted  in  the  sea  urchin  egg  by  several  investi- 
gators. 

Such  changes  have  recently  been  more  carefully 
investigated  by  Heilbrunn  (191 5)  and  by  Chambers 
(191 7).  Heilbrunn  tested  the  viscosity  of  the  proto- 
plasm of  the  egg  of  the  sea  urchin  by  the  use  of  the 
centrifuge,  and  found  that  the  separation  of  protoplasmic 
granules  of  different  specific  gravity  by  centrifugal  force  is 
much  more  readily  effected  before  than  after  fertilization. 
The  protoplasm  is  much  more  fluid  before  than  after 
fertilization,  and  this  can  mean  only  a  tendency  toward 
gelation  in  a  colloidal  system  such  as  protoplasm;  this 
change  was  observed  to  begin  two  and  one-half  minutes 
after  insemination.  Heilbrunn  also  determined  that  a 
variety  of  parthenogenetic  agents,  and  he  believes  all  such 
agents,  produce  a  similar  gelation  effect,  and  he  was 
therefore  led  to  regard  the  coagulation  change  as  the 
primary  event  in  activation  of  the  egg. 

Chambers  investigated  this  subject  by  means  of  the 
micro-dissection  method  as  part  of  a  study  of  the  cell 
aster  which  he  interprets  as  a  reversible  gelation  phe- 
nomenon. When  the  sperm  aster  appears  in  the  egg 
of  Echinarachnius  about  three  minutes  after  penetra- 
tion of  the  spermatozoon,  it  can  be  shown  to  be  a  more 
rigid  area  by  the  fact  that  it  can  be  pushed  and  rolled 
in  the  surrounding  more  liquid  cytoplasm  by  the  micro- 
dissecting  needle.  As  the  aster  increases  in  size  the 
gelation  effect  extends.  Chambers  thus  localizes  the 
gelation  due  to  fertilization  in  the  sperm  aster,  though 
Heilbrunn's  results  appear  to  indicate  a  more  general 
effect;    there  is,  however,  nothing  in  Chamber's  result 


154  PROBLEJMS  OF  FERTILIZATION 

inconsistent  with  the  idea  of  a  general  gelation  effect, 
which  is  decidedly  more  pronounced  in  the  region  of 
the  aster.  Karyokinesis  is,  according  to  Chambers, 
essentially  a  reversible  gelation  effect.  The  gel  area 
represented  by  the  sperm  aster  is  a  preparation  for  the 
first  cleavage  of  the  egg. 

4.  Chemical  changes. — The  initiation  of  development 
which  is  associated  with  fertilization  involves  a  progres- 
sive chain  of  chemical  changes,  but  it  is  obvious  that 
these  lead  us  outside  of  the  field  of  fertilization  proper, 
beyond  the  stage  of  indicia  of  fertilization.  There  is, 
however,  one  very  striking  change  occurring  at  the 
very  beginning  of  the  fertilization  reactions  that  de- 
serves notice  here.  I  allude  to  the  complete  loss  of 
sperm-agglutinating  substance  by  the  egg.  This  was 
first  observed  by  the  writer  in  the  case  of  Nereis,  and 
was  subsequently  more  carefully  studied  in  Arhacia 
(1914-15).  As  long  as  eggs  of  Arhacia  retain  capacity  for 
fertilization  they  produce  this  substance,  which  is  read- 
ily recognizable  by  its  agglutinating  action  on  sperm 
suspensions  of  the  same  species.  But  if  the  adherent 
jelly  which  also  contains  this  substance  is  removed  from 
fertilized  eggs  and  the  substance  present  before  fertili- 
zation is  washed  off,  not  a  trace  of  the  agglutinating 
substance  can  be  derived  from  the  fertilized  eggs. 
There  is  a  complete  loss  of  sperm-agglutinating  sub- 
stance and  with  it  a  loss  of  capacity  for  further  fertili- 
zation reaction  at  the  moment  of  fertilization.  This 
determination  has  been  confirmed  by  Glaser  (19 14), 
by  Just  (19 19),  and  by  Moore  (1916).  The  writer  was 
convinced  by  this  and  other  data,  discussed  later,  that 
the  agglutinating  substance  is  necessary  for  fertilization. 


IJIE  niYSIOLOGY  OF  FERTILIZATION  icc 

Its  loss  therefore  constitutes  a  very  striking  chemical 
change  directly  due  to  fertilization.  A  similar  loss  follows 
the  effective  action  of  parthenogenetic  agents  which 
render  the  eggs  insusceptible  to  fertilization  (Lillie,  19 14; 
Moore,  1916). 

IV.    PARTIAL   FERTILIZATION 

As  fertilization  is  a  series  of  processes  it  should  be 
possible  to  arrest  the  series  at  various  stages  and  thus 
to  discover  the  value  of  the  various  steps  for  develop- 
ment. The  experiments  which  have  been  directed  to- 
ward this  end  are  not  very  numerous,  as  considerable 
difficulties  exist  in  arresting  the  progress  of  fertihzation 
without  destroying  the  entire  zygote. 

The  writer  (1911-12)  found  ideal  material  for  solu- 
tion of  part  of  this  problem  in  the  egg  of  Nereis.  As 
we  have  previously  seen,  the  spermatozoon  produces 
the  cortical  changes  before  its  penetration  into  this  egg, 
which  does  not  occur  until  about  forty-five  minutes 
after  initiation  of  the  cortical  changes.  During  this 
time  it  is  imbedded  in  the  jelly  secreted  by  the  egg. 
If  the  egg  be  centrifuged  at  the  proper  time,  the  jelly, 
which  is  of  much  less  specific  gravity  than  the  egg, 
separates  from  it  completely,  and  in  so  doing  frequently 
removes  the  spermatozoon.  That  the  centrifuging  pro- 
cess itself  is  not  deadly  is  proved  by  the  fact  that  those 
eggs  from  which  the  spermatozoon  is  not  removed 
always  segment  and  may  develop  approximately  nor- 
mally. 

The  eggs  from  which  the  spermatozoon  is  removed 
complete  the  maturation  process  which  is  initiated  by 
the  spermatozoon,  but  they  never  segment.     The  eg<^ 


156  PROBLEMS  OF  FERTILIZATION 

nucleus  (female  pronucleus)  arises  and  attains  the  same 
size  as  in  normally  fertilized  eggs.  The  chromosomes 
of  the  first  cleavage  spindle  then  form  in  the  usual 
fashion  and  at  the  usual  time,  accompanied  by  dis- 
appearance of  the  nuclear  membrane.  But,  whereas 
in  the  presence  of  a  sperm  nucleus  cytoplasmic  asters 
accompany  these  changes  and  a  spindle  rapidly  arises, 
in  the  absence  of  the  sperm  nucleus  there  is  absolutely 
no  sign  of  cytasters  or  evidence  of  spindle  formation. 
The  chromosomes  lie  naked  in  the  cytoplasm,  surrounded 
by  a  clear  area.  Each  chromosome  then  splits  longitu- 
dinally in  the  usual  fashion,  but  the  halves  do  not  sepa- 
rate. At  the  time  of  the  telophase  of  the  normal  first 
cleavage  there  is  a  tendency  to  scattering  and  breaking 
up  of  the  chromosomes.  When  the  normal  eggs  have 
reached  the  two-  and  four-celled  stages,  the  scattering 
and  breaking  up  of  the  chromosomes  have  progressed 
much  farther  in  the  unsegmented  eggs,  and  in  the 
course  of  two  or  three  hours  there  remains  no  differ- 
entiated nucleus  or  chromosomes,  but  only  numerous 
chromatic  granules  scattered  throughout  the  cyto- 
plasm. 

This  experiment  then  shows  that  the  fertiHzation 
processes  may  be  divided  physiologically  as  well  as 
morphologically  into  the  two  main  phases  of  the  exter- 
nal and  internal  phenomena.  The  external  action  is 
adequate  to  produce  the  cortical  changes  alone,  but  not 
the  entire  series  of  developmental  events.  In  most  eggs 
the  penetration  of  the  spermatozoon  is  so  rapid  that  it 
accompanies  or  precedes  the  cortical  changes,  and  it 
is  therefore  difficult  to  ascertain  whether  the  cortical 
changes  are  dependent  on  penetration  or  not.     Loeb 


THE  PHYSIOLOGY  OF  FERTILIZATION  157 

(1913)  has,  however,  been  able  to  show,  by  means  of 
hybridization  experiments,  that  the  cortical  changes  in 
the  sea  urchin  are  not  dependent  on  penetration.  He 
found  that  the  sperm  of  Asterias  would  cause  the  for- 
mation of  fertilization  membranes  in  the  eggs  of  Slron- 
gylocentrotus  purpuratus  in  hyperalkahne  sea-water,  but 
only  a  fraction  of  them  segmented.  Cytological  obser- 
vation showed  that  the  spermatozoon  penetrated  only 
a  similar  fraction,  presumably  the  ones  that  segmented. 
In  others  there  was  external  action  of  the  spermatozoon 
alone.  The  eggs  which  had  not  been  entered  by  the 
spermatozoon  could  be  caused  to  develop  by  treatment 
with  hypertonic  sea-water.  It  is  therefore  probable 
that  it  is  a  general  rule  of  fertilization  that  the  sperma- 
tozoon sets  the  cortical  changes  of  the  egg  in  operation 
before  penetration. 

Similarly  the  activation  of  the  egg  by  means  of  parthe- 
nogenetic  agents  has  been  shown  in  certain  cases  to  be 
clearly  divisible  into  two  phases.  This  is  shown  most 
clearly  by  Loeb's  famous  method  of  inducing  develop- 
ment in  sea  urchin  eggs,  which  consisted  of  a  brief 
treatment  with  butyric  acid  or  some  other  cytolytic 
agency,  producing  the  cortical  changes  alone,  which 
required  to  be  supplemented  by  after-treatment  with 
hypertonic  sea-water  or  certain  other  agencies  to  induce 
complete  development.  Loeb  explained  this  result  b}' 
supposing  that  the  cortical  changes  in  themselves  have 
something  of  excess  in  them,  leaving  the  egg  in  a  sickly 
condition,  and  that  this  effect  was  counteracted  by  the 
second  treatment.  Whether  this  is  a  correct  explanation 
or  not  we  need  not  now  inquire  (see  chap,  vii),  but  we 
should  note  that  the  transition  from  the  first  to  the 


158  PROBLEMS  OF  FERTILIZATION 

second  phase  is  much  more  gradual  in  most  forms  than 
it  is  in  the  sea  urchins  and  usually  gives  the  impression 
of  being  a  continuous  process. 

We  have  seen  previously  that  the  spermatozoon 
bears  a  substance  essential  for  fertilization,  which  is 
readily  lost.  We  may  therefore  suppose  that  it  is  this 
substance  which  activates  the  cortical  changes  of  the  egg. 

The  questions  may  now  be  raised:  (i)  How  soon 
after  entrance  of  the  spermatozoon  into  the  egg  is  the 
activation  process  completed?  (2)  Is  there  a  quanti- 
tative relation  in  the  internal  phenomena  of  activation  ? 
Wilson  (1903)  observed  that  if  the  fertilized  eggs  of 
Cerehratulus  be  cut  in  two  shortly  after  the  penetration 
of  the  spermatozoon  in  such  a  way  that  one  part  con- 
tains the  sperm  nucleus  and  the  other  the  egg  nucleus, 
only  the  former  develops,  while  the  part  containing  the 
egg  nucleus  completes  its  maturation  but  goes  no  farther; 
it  was  therefore  incompletely  fertihzed  at  this  time. 
Ziegler  (1898)  observed  that  if  the  egg  of  the  sea  urchin 
be  so  constricted  after  fertilization  that  one  part  con- 
tains the  sperm  nucleus  and  the  other  the  egg  nucleus, 
the  part  that  contains  the  sperm  nucleus  undergoes 
cleavage  and  develops  farther;  in  the  other  part  the 
egg  nucleus  undergoes  remarkable  transformations,  dis- 
solving and  reappearing,  a  process  which  is  repeated 
several  times,  but  this  part  does  not  undergo  cleavage. 
In  spite,  therefore,  of  the  presence  of  the  sperm  nucleus 
in  a  constricted  portion  of  the  same  egg,  the  part  con- 
taining the  egg  nucleus  was  not  completely  fertilized. 
Yet  we  know  from  experiments  on  artificial  partheno- 
genesis that  the  egg  nucleus  is  capable  of  complete 
activation  without  participation  of  the  sperm  nucleus. 


THE  PHYSIOLOGY  OF  FERTH^IZA TION  159 

Boveri  (1895)  has  also  observed  that,  if  freshly  ferti- 
lized sea  urchin  eggs  be  broken  into  fragments  by  shak- 
ing, those  fragments  which  contain  the  egg  nucleus 
alone  do  not  usually  segment,  though  the  nucleus  en- 
larges, dissolves,  and  reappears,  but  some  such  pieces 
may  segment  once  or  twice  and  then  stop  (1902). 

It  is  evident,  therefore,  that  the  sperm  sets  in  opera- 
tion a  progressive  series  of  processes  within  the  egg 
and  that  complete  activation  is  not  attained  by  any 
means  at  once  after  entrance  of  the  spermatozoon,  and 
probably  not  until  about  the  time  of  union  of  egg  and 
sperm  nuclei. 

The  question  arises  whether  this  is  a  purely  quanti- 
tative relation  or  whether  the  spermatozoon  is  involved 
in  a  series  of  quahtatively  different  processes  each  of 
which  requires  its  aid  or  other  extraneous  support. 
There  is  at  present  no  evidence  for  the  latter  concep- 
tion. On  the  quantitative  side  we  would  have  the  two 
questions,  whether  the  sperm  activates  by  means  of  a 
substance  which  it  slowly  releases,  or  whether  it  acti- 
vates a  substance,  or  ferment-like  bodies,  contained 
within  the  egg.  As  Loeb  has  pointed  out,  if  the  first 
assumption  is  correct  we  would  expect  that  two  sper- 
matozoa would  cause  a  more  rapid  progress  of  events 
within  the  egg  than  a  single  spermatozoon;  but  this  is 
not  the  case;  dispermic  eggs  segment  in  the  same 
tempo  as  monospermic  eggs. 

The  writer  (191 2)  has  observed  that  portions  of 
sperm  nuclei  obtained  by  centrifuging  Nereis  eggs  dur- 
ing fertilization  before  penetration  of  the  spermatozoon 
produce  an  effect  in  terms  of  the  sperm  aster  roughly 
proportional  to  their  size;    this  can  be  understood  in 


i6o  PROBLEMS  OF  FERTILIZATION 

either  of  the  foregoing  senses;  but  there  is  apparently 
no  slackening  in  rate  of  the  processes,  which  merely 
occur  on  a  smaller  scale  or  with  less  energy.  Theoret- 
ically such  experiments  should  enable  one  to  determine 
how  much  of  the  spermatozoon  is  essential  for  complete 
activation,  but  the  practical  difficulties  have  so  far 
prevented  such  a  determination. 

The  quantitative  side  of  fertilization  furnishes  a 
very  interesting  problem.  It  would  seem  that  incom- 
plete activation  of  the  egg  is  not  compensated  in  later 
stages  but  sooner  or  later  results  in  a  complete  arrest 
of  the  vital  machinery. 

It  is  possible  that  we  should  also  include  in  the 
conception  of  partial  fertilization  cases  of  fertilization 
with  stale  or  injured  gametes;  if  either  the  egg  or  sper- 
matozoon be  involved  development  takes  a  slower 
tempo  and  ceases  sooner  or  later  with  development  of 
various  abnormalities.  Such  conditions  may  obviously 
grade  all  the  way  up  to  the  normal  (see  Stockard, 
Dungay,  Hertwig).  Such  results  should  not,  however, 
be  understood  in  a  purely  quantitative  sense,  but  also 
in  the  sense  of  a  growing  disharmony  dependent  on 
variation  in  the  degree  of  the  effect  upon  different 
portions  of  the  egg. 

In  the  case  of  the  artificial  induction  of  partheno- 
genesis the  quantitative  aspect  of  activation  has  been 
very  accurately  measured  by  R.  S.  Lillie  (19 15)  in  the 
case  of  the  starfish  egg.  Under  the  influence  of  higher 
temperatures  (about  29-36°  C.)  or  butyric  acid  (about 
N/260  concentration)  the  activation  process  is  started 
in  the  egg.  If  the  process  is  allowed  to  proceed  to  a 
certain  optimum  stage  and  the  eggs  are  then  returned 


THE  PHYSIOLOGY  OF  FERTILIZATION  i6i 

to  sea-water  of  normal  temperature  and  composition, 
the  eggs  develop  normally  to  the  formation  of  larvae. 
If,  however,  the  process  is  interrupted  too  soon,  the  egg 
is  able  only  to  begin  the  first  processes;  but  within  a 
suitable  period  of  time  renewal  of  the  action  to  the 
optimum  extent  will  bring  about  complete  activation. 
The  activation  process  can  thus  be  arbitrarily  inter- 
rupted and  resumed,  and  this  with  either  high  tempera- 
ture or  butyric  acid  alone,  or  with  combination  of  the 
two  in  either  order.  The  optimum  time  of  exposure 
to  high  temperature  shows  a  very  high  temperature 
coefficient  (Qio  =  200-400) ,  but  the  quantitative  details 
do  not  concern  us  here  (see  chap.  vii). 

V.    POLYSPERMY   AND   MEROGONY — THE   PROBLEM   OF 

REVERSIBILITY 

The  fertilized  egg  does  not  react  to  fresh  spermat- 
ozoa, and  these  do  not  enter  it.  The  change  which 
produces  this  condition  takes  place  so  rapidly  that  only 
one  spermatozoon  normally  enters,  though  hundreds 
may  be  present  in  the  immediate  neighborhood  of  the 
egg,  and  many  may  reach  it  apparently  simultaneously. 
The  fact  of  monospermy  has  been  known  since  the  time 
of  the  classic  researches  of  Hertwig  and  of  Fol.  As  is 
well  known,  Fol  proposed  the  theory  that  the  fertili- 
zation membrane  prevented  entrance  of  supernumerary 
spermatozoa  and  constituted  the  mechanism  for  pre- 
vention of  polyspermy.  Its  formation  is,  however, 
much  too  slow  to  account  for  the  facts;  and  it  has  been 
repeatedly  shown  that  removal  of  the  fertilization  mem- 
brane does  not  render  the  egg  more  susceptible  to 
superimposed  inseriijnation. 


i62  PROBLEMS  OF  FERTILIZATION 

The  mechanism  for  the  prevention  of  polyspermy 
must  operate  at  an  exceedingly  high  rate  of  speed; 
this  is  readily  appreciated  when  we  consider  that  it 
is  effective  in  selecting  only  one  out  of  the  many  sper- 
matozoa that  appear  to  the  eye  to  reach  the  ovum 
simultaneously.  It  must  therefore  be  effective  before 
the  fertilization  membrane  forms,  so  that  it  becomes 
somewhat  superfluous  to  inquire  whether  this  mem- 
brane furnishes  an  unnecessary  further  protection. 
This  subject  is  discussed  further  in  chapter  vii. 

An  excellent  method  for  experimental  investigation 
of  this  problem  was  discovered  by  0.  and  R.  Her  twig 
(1887),  who  found  that  when  sea  urchin  eggs  are  broken 
into  fragments  the  non-nucleated  fragments  may  be 
fertilized  no  less  than  the  nucleated  fragments,  a  phe- 
nomenon that  subsequently  became  known  as  me- 
rogony.  Boveri  (1889),  Morgan  (1895),  ^^d  Seeliger 
(1894-96)  utilized  this  method  for  study  of  the  problem 
of  nuclear  transmission  of  hereditary  qualities;  Driesch 
and  others  for  problems  of  physiology  of  development; 
and  the  method  was  subsequently  used  for  testing  the 
changes  in  the  cytoplasm  of  the  egg  with  reference  to 
fertilizability. 

The  classic  experiments  along  the  latter  lines  are 
those  of  Delage  (1898,  1899,  1901)  and  of  E.  B.  Wilson 
(1903).  Delage's  observations  included  sea  urchins, 
starfish,  an  annelid  (Lanicc),  and  a  mollusk  {Dentalium). 
Fragments  taken  from  eggs  with  intact  germinal  vesicle 
are  unfertilizable;  but  fragments  taken  from  eggs  in 
which  the  germinal  vesicle  has  begun  to  break  down 
may  fertilize,  and  after  complete  dissolution  of  the 
germinal  vesicle  fragments  are  completely  fertilizable. 


THE  niYSIOLOGY  OF  FERTILIZATION  163 

In  the  starfish  this  condition  lasts  until  after  the  forma- 
tion of  the  first  polar  globule,  but  when  the  second 
polar  body  begins  to  appear  the  fragments  are  less 
fertilizable,  even  if  they  do  not  become  completely  resist- 
ant to  merogonic  fertilization.  Wilson  made  observa- 
tions similar  in  many  respects  in  the  case  of  Cerebraiuliis, 
and  demonstrated  in  addition  that  fragments  of  ferti- 
lized eggs  are  completely  unfertilizable.  Such  fragments 
have  a  fresh-cut  surface  and  entirely  lack  any  me- 
chanical protection. 

It  will  be  observed  that  the  fertilizable  character 
of  the  egg  bears  no  relation  to  morphological  membranes 
in  these  cases,  for  in  all  we  are  dealing  with  freshly  cut 
pieces  with  an  exposed  surface.  The  egg  passes  from  a 
non-fertihzable  to  a  fertilizable  condition,  and  after 
fertilization  to  a  non-fertilizable  condition  again.  A 
fragment  of  a  fertilized  egg,  devoid  of  the  sperm  nu- 
cleus, does  not  return  to  a  fertihzable  condition.  There 
is  no  indication  that  the  processes  so  far  are  reversible. 

We  may  express  these  results  by  saying  that  the  egg 
has  acquired  a  physiological  protection,  as  contrasted 
with  the  earher  assumed  mechanical  protection,  against 
polyspermy. 

Superposition  of  fertilization  on  parthenogenesis.— 1\\- 
asmuch  as  fertilization  and  activation  of  the  egg  bv 
parthenogenetic  agents  produce  similar  morphological 
and  physiological  results  on  the  whole,  it  would  seem 
that  both  methods  should  produce  eggs  equally  resistant 
to  superimposed  fertilization.  If  this  were  not  the  case 
it  would  seem  that  artificial  activation  is  not  the  full 
equivalent  of  fertilization  with  reference  to  initiation 
of   development  in  spite  of  the  similarity  of   results 


1 64  PROBLEMS  OF  FERTILIZATION 

attained,  or  that  the  process  of  artificial  activation, 
unlike  normal  fertilization,  is  reversible.  The  first 
alternative  runs  counter  to  all  the  physiological  deter- 
minations, and  the  second  is  of  so  fundamental  a  character 
that  it  requires  most  careful  analysis. 

Loeb   (19 1 3)   found  that  eggs  of  Strongylocentrotus 
purpuratuSj   in  which  membrane  formation  had   been 
induced  by  butyric  acid,  could  be  fertihzed  by  sperm 
if  the  membrane  were  torn  by  shaking;    they  differed 
therefore  in  this  respect  from  eggs  in  which  membrane 
formation  had  been  induced  by  f ertihzation ;    but  it  is 
noteworthy  that  such  eggs  differ  from  normally  ferti- 
lized eggs  in  requiring  a  second  treatment  after  mem- 
brane formation  to  induce  development,  so  that  the 
activation  by  butyric  acid  is  incomplete.     Loeb  (1913) 
also   determined    that   eggs    of   the    same    form   when 
treated  with  hypertonic  sea-water  alone  might  begin 
development,  but  some  of  them  come  to  a  standstill 
in  the  2-,  4-,  8-,  or  i6-celled  stage,  and  that  such  eggs 
were  fertilizable  in  the  sense  that  insemination  may  cause 
the  formation  of  a  separate  membrane  around  each  blas- 
tomere;    they  then  resume  development  in  a  perfectly 
normal   way,  according  to  Loeb,  and  become   normal 
larvae.     Here  again  fertilization  is  superimposed  upon 
incomplete  parthenogenesis.     Loeb  concluded  merely  in 
connection  with  these  experiments  that  the  block  to 
polyspermy  is  not  due  to  the  changes  necessarily  con- 
nected with  development. 

In  later  papers  (1913^  and  191 5)  he  found  that 
9,lkalies  induce  development  of  eggs  of  Arhacia  in  a 
manner  somewhat  similar  to  that  of  butyric  acid;  but 
if  the  ^ggs  after  treatment  for  the  proper  length  of  time 


THE  PHYSIOLOGY  OF  FERTILIZATION  i6s 

are  put  into  a  solution  which  prevents  their  develop- 
ment (sea-water  with  chloral  hydrate  or  NaCN)  when 
taken  out  they  behave  as  though  nothing  had  happened 
to  them.     He  considers  this  a  demonstration  that  arti- 
ficial activation  can  be  reversed.     Now  it  is  notable 
that  in  such  treatment  no  visible  change  occurs  in  the 
alkali,  but  only  after  transfer  to  sea- water.     It  would 
seem  then  to  be  a  reasonable  interpretation  that  such 
changes  are  prevented  by  the  chloral  or  NaCN.     What 
is  reversed  therefore  in  this  case  is  at  most  a  condition 
which  permits  of  cortical  changes  in  sea-water.     If  the 
eggs  are  placed  from  the  alkali  in  sea-water,  even  if 
only  for  a  few  minutes,  before  the  chloral  or  NaCN 
sea-water,  they  will  not  ''reverse." 

It  is  apparent  that  such  results  can  be  as  readily 
understood  in  the  quantitative  sense  of  partial  or  arrested 
activation  as  in  terms  of  reversal.  The  fertilization 
reaction  has  definite  quantitative  relations,  as  we  have 
seen  in  discussing  partial  fertilization;  the  same  is  true 
of  any  part  of  the  series  of  reactions,  and  it  certainly 
holds  for  the  cortical  reactions  in  activation.  If,  there- 
fore, the  cortical  reactions  are  incomplete  in  any  experi- 
ment the  possibility  of  superimposing  fertihzation  on 
such  incomplete  reactions  might  remain. 

C.  R.  Moore  (1916)  has  shown  in  experiments  con- 
ducted under  the  writer's  observation  that  such  a  quan- 
titative relation  actually  obtains.  To  understand  the 
experiments  and  results  it  is  essential  to  examine  the  char- 
acter of  artificial  activation  by  means  of  butyric  acid, 
which  was  the  agent  used  in  both  Loeb's  and  Moore's  ex- 
periments. The  strength  of  the  acid  and  time  of  exposure 
are  variable  factors.    Fifty  c.c.  sea-water  -\-2  ,S  ex.  N/io 


i66  PROBLEMS  OF  FERTILIZATION 

butyric  acid  is  a  very  favorable  strength  for  artificial 
activation;  it  was  therefore  used  constantly  in  these 
experiments.  Now  it  must  be  emphasized  that  there 
is  no  visible  change  in  the  eggs  while  in  the  butyric 
acid;  but  wheh  the  eggs  are  transferred  to  sea-water, 
if  the  time  of  exposure  has  been  right,  the  eggs  form 
membranes  as  they  do  after  insemination,  and  sub- 
sequent treatment  with  h}^ertonic  sea-water  will  cause 
them  to  develop  normally.  By  varying  the  time  of 
exposure  to  the  butyric  acid  we  get  varying  degrees  of 
activation  as  expressed  in  membrane  formation  by  the 
eggs  and  in  their  capacity  for  development;  this  pro- 
ceeds up  to  an  optimum.  If  the  exposure  to  butyric 
acid  be  prolonged  beyond  the  optimum  the  capacity  for 
development  gradually  falls  off  to  zero. 

It  is  clear  that  butyric  acid  produces  a  condition  of 
pre-activation  in  which  the  egg  activates  when  returned 
to  its  normal  environment;  but  if  the  treatment  be  too 
short  the  pre-activation  is  insufficient;  if  it  be  too  long 
some  other  condition  arises  that  inhibits  normal  acti- 
vation. 

If,  then,  insemination  is  practiced  on  these  eggs 
after  their  return  to  sea-water,  it  is  found  to  be  success- 
ful in  inverse  proportion  to  the  degree  of  activation 
induced  in  the  sea-water  up  to  the  optimum  point,  at 
which  superimposed  insemination  has  no  effect.  Be- 
yond this  the  curve  of  superimposed  fertilization  shows 
a  second  rise  and  fall  to  zero.  At  the  optimum  point 
for  artificial  activation,  at  which  all  eggs  form  mem- 
branes and  are  capable  of  development  after  treatment 
by  hypertonic  sea-water,  the  sperm  has  absolutely  no 
fertilizing  effect  on  the  eggs,  whatever  its  concentra- 


THE  PHYSIOLOGY  OF  FERTILIZAIIOX  167 

tion,  even  if  the  membranes  formed  by  the  butyric 
acid  be  entirely  destroyed.  Such  eggs  are  therefore 
comparable  to  normally  fertilized  eggs  in  respect  to 
their  unfertilizable  condition.  Moore  has  shown  that, 
if  they  be  very  heavily  inseminated,  spermatozoa  may 
enter  them,  but  there  is  absolutely  no  reaction  between 
sperm  and  egg. 

Thus  in  the  case  of  Arbacia,  on  which  these 
experiments  were  performed,  there  is  no  possibility  of 
superimposing  fertilization  upon  parthenogenesis  after 
optimum  exposure  to  the  activating  agent.  But  if  the 
exposure  to  the  activating  agent  be  too  short,  or  too 
long,  some  degree  of  capacity  for  fertilization  exists, 
which  is  expressed  in  Figs,  iga,  b.  But  development 
due  to  insemination  after  too  long  exposure  to  butyric  acid 
is  never  normal.  It  is  obvious  that  we  are  dealing  here 
with  a  quantitative  relation  that  would  be  as  true  for  each 
egg  as  for  the  entire  culture.  The  individual  variability 
of  the  eggs  is  what  gives  the  percentage  results. 

If  we  were  to  assume  the  presence  of  a  single  activ- 
able  substance  within  the  egg  for  w^hich  the  sper- 
matozoa have  a  certain  alHnity  the  results  concerning 
superposition  of  fertihzation  on  parthenogenesis  could 
be  expressed  as  follows:  Shorter  exposure  to  the  arti- 
ficial activating  agents  leaves  varying  amounts  of  the 
activable  substance  unengaged  for  sperm  action;  the 
optimum  exposure  leaves  none  free;  too  long  action 
leaves  some  unengaged,  owing  to  secondary  conditions 
produced  in  the  pre-activation  period,  which  hamper 
the  development  after  insemination.  Moore  has  shown 
that  there  is  a  close  approximation  in  the  content  of 
the  sperm-agglutinating  substance  of  the  eggs  to  this 


1 68 


PROBLEMS  OF  FERTILIZATION 


hypothesis   and   has   therefore   confirmed   the   writer's 
opinion  that  this  is  the  activable  substance  of  the  egg. 

The  same  author  (Moore,  191 7)  has  also  examined 
Loeb's  statement  concerning  the  fertilizabihty  of  eggs 
treated  by  hypertonic  sea-water  with  results  concordant 


10   O   »')   o 

M     CO    Tt    \0 


100 

90 
80 
70 
60 

50 
40 
30 
20 
10 


1 00" 

90 
80 
70 
60 

so 


■^OtoOvoO'oO         loO         loOvoO         10        O 


a 


o 

CO 
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0 

00 

't 

■* 

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00 


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to    ^O 


Fig.  19. — Curve  of  fertilization  superimposed  upon  butyric  add 
treatment  in  Arbacia.  Curve  b  continues  curve  a.  The  ordinates 
represent  percentages  of  fertilization  as  measured  by  cleavage  and  the 
abscissae  length  of  previous  exposure  to  butyric  acid  in  seconds  (after 
C.  R.  Moore). 


THE  PHYSIOLOGY  OF  FERTILIZATION  169 

with  the  above:  ''The  superposition  of  insemination 
on  the  optimum  hypertonic  treatment  does  not  in- 
crease the  percentage  of  development."  This  is  in  no 
sense  inconsistent  with  Loeb's  statement  that  the  block 
to  polyspermy  is  not  due  to  changes  necessarily  con- 
nected with  development,  whatever  this  may  mean; 
but  it  is  a  result  that  renders  in  the  highest  degree 
improbable  that  reversal  of  activation  of  the  egg  occurs. 
Activation  is  a  part  of  the  fertilization  reactions,  and 
there  remains  no  evidence  that  it  is  reversible,  whether 
induced  by  fertihzation  or  by  artificial  means. 

Godlewski  (191 2)  has  shown  that  the  sperm  of 
Chaetopterus  will  enter  the  eggs  of  sea  urchins  and  cause 
normal  membrane  formation;  but  such  eggs  do  not 
segment,  and  soon  die.  If,  however,  they  are  exposed 
after  the  hybrid  fertilization  to  the  action  of  hypertonic 
sea-water  for  a  short  period  of  time,  they  may  segment 
regularly  and  develop  to  larvae.  He  speaks  of  this  as 
superposition  of  parthenogenesis  on  hybrid  fertilization. 
This  determination  in  no  way  runs  counter  to  our  inter- 
pretation, however  it  may  be  named.  It  is  quite  clear 
that  the  hypertonic  sea-water  has  no  activating  effect 
in  this  experiment,  but  on  the  contrary  inhibits  an  in- 
jurious effect  of  the  foreign  sperm. 

Herbst's  experiments  (1909  and  191 2),  in  which  he 
superimposed  hybrid  fertilization  on  partial  partheno- 
genetic  activation  and  secured  a  shifting  of  inheritance 
in  the  maternal  direction,  deal  primarily  with  problems 
of  hybridization.  As  far  as  the  problem  of  superposition 
is  concerned  the  experiments  are  perfectly  consistent 
with  JVIoore's  interpretation,  because  the  parthenogenetic 
activation  was  incomplete. 


lyo  PROBLEMS  OF  FERTILIZATION 

VI.    EXTERNAL   CONDITIONS    OF   FERTILIZATION 

The  fertilization  reactions  that  we  have  been  con- 
sidering are  dependent,  not  only  on  the  internal  condi- 
tions of  the  gametes,  but  also  on  the  nature  of  the 
medium  in  which  insemination  occurs.  Fertilization 
always  occurs  in  an  aqueous  medium  containing  a  bal- 
anced solution  of  salts,  of  which  NaCl,  MgCl,,  KCl,  and 
CaCL  are  the  chief.  In  such  a  medium  fertiHzation 
depends,  within  the  usual  range  of  temperature,  on  re- 
action of  the  medium  (acidity  or  alkalinity)  and  the 
balance  and  concentration  of  the  salts.  Sea-water  is  a 
medium  of  this  kind  in  which  the  variable  factors  can 
be  readily  controlled.  Most  studies  have  therefore  been 
made  on  marine  animals;  but  there  is  abundant  evidence 
that  the  same  principles  apply  to  other  animals. 

The  effect  of  reaction  of  the  medium  may  be  shown 
by  some  hitherto  unpublished  experiments  on  starfish 
eggs  made  in  1914.  In  the  summer  these  eggs  (at 
Woods  Hole)  frequently  do  not  fertilize  very  readily 
in  normal  sea-water,  but  they  may  be  made  to  do  so 
by  a  slight  increase  in  the  alkalinity;  on  the  other 
hand  an  increase  of  acidity  tends  to  inhibit  fertilization; 
the  following  experiment  demonstrates  the  principle.  A 
series  of  eleven  watch  glasses  is  laid  out  in  the  order  i 
to  II,  of  which  the  center  one  (No.  6)  contains  normal 
sea-water,  those  to  the  left  increasing  concentrations  of 
HCl,  and  those  to  the  right  increasing  concentra- 
tions of  NaOH  in  sea-water  as  follows:  No.  1,  N/$oo 
HCl;  No.  2,  iV/i,ooo  HCl;  No.  3,  AV2,ooo  HCl;  No.  4, 
A/4,000  HCl;  No.  5,  A/10,000  HCl;  No.  6,  normal  sea- 
water;  No.  7,  A/10,000  NaOH;  No.  8,  A/4,000  NaOH; 
No.  9,  A/2,000  NaOH;  No.  10,  A/1,000  NaOH;  No.  11, 


THE  PHYSIOLOGY  OF  FERI ILIZATION  171 

iV"/5oo  NaOH.  The  same  quantity  of  a  single  lot  of  eggs 
was  then  added  to  each  crystal  and  was  fertilized  with 
the  same  quantity  of  a  single  lot  of  sperm.  The  percent- 
age of  eggs  that  segmented  in  each  dish  were  as  follows: 

Glass  No. 

123456789  10  II 

Percentage 

o       2.5       8     22.5     45       53       84     92.5     88.5     89  o 

In  the  highest  concentration  of  acid  the  unsegmented 
eggs  were  devoid  of  fertilization  membranes,  thus  unfer- 
tilized, but  in  the  highest  concentrations  of  the  alkali 
the  unsegmented  eggs  had  membranes  and  were  thus 
fertiHzed,  but  cleavage  was  inhibited  by  the  OH  ions. 

It  will  be  observed  that  the  curve  derived  from  the 
percentages  of  eggs  fertilized  ascends  regularly  from  the 
acid  to  the  alkaline  end  of  the  series.  Very  striking 
demonstrations  of  the  favoring  effects  of  NaOH  on 
fertilization  may  be  obtained  from  eggs  that  give  only 
a  very  low  percentage  of  fertilization  in  normal  sea- 
water,  but  may  give  a  very  high  percentage  in  alkalized 
sea-water. 

The  presence  of  alkali  favors  the  reaction  between 
the  egg  and  spermatozoon,  probably  because  it  tends 
to  make  the  plasma  membrane  permeable  and  thus  to 
permit  a  closer  relation  between  the  activable  substance 
of  the  egg  and  the  activating  substance  of  the  spermato- 
zoon; this  favors  entrance  of  the  spermatozoon  into 
the  egg.  The  use  of  a  hyperalkaline  medium  to  facili- 
tate fertilization  was  first  made  by  Loeb  in  a  successful 
attempt  to  produce  heterogeneous  hybridization,  and 
has  since  been  widely  extended  for  the  same  purpose. 
It  should  be  noted  that  the  reaction  is  favored  only  in 
the  actual  presence  of  the  alkali;  previous  treatment  of 


172  PROBLEMS  OF  FERTILIZATION 

eggs  alone,  of  sperm  alone,  or  of  both,  before  insemina- 
tion in  normal  sea-water  does  not  increase  the  percent- 
age of  fertilization.  We  are  therefore  probably  dealing 
with  a  rapidly  reversible  modification  of  the  surface  of 
one  or  both  kinds  of  gametes. 

With  reference  to  the  question  of  balance  of  salts, 
the  only  systematic  experiments  are  those  of  Loeb 
(1914,  1915^),  who  found  that,  for  the  fertilization  of 
eggs  of  the  sea  urchin,  the  presence  of  Ca  and  OH  ions 
is  very  important.  Eggs  and  sperm  washed  in  neutral 
7V/2  NaCl  will  not  fertilize  in  this  salt  alone,  nor  in 
combinations  of  two  or  more  of  NaCl,  MgCla,  and  KCl 
in  the  proportions  and  concentrations  in  which  these 
salts  exist  in  sea-water,  though  the  spermatozoa  may 
be  very  active  and  fill  the  jelly  of  the  eggs.  But  the 
addition  of  CaCl2  to  NaCl,  or  to  NaCl  and  MgCL,  or 
to  NaCl  and  MgCla  and  KCl  in  the  sea-water  propor- 
tions will  induce  normal  fertihzation;  this  will  happen 
even  more  promptly  and  certainly  if  a  little  NaOH  is 
added  at  the  same  time.  Loeb  states  (1914)  that  cal- 
cium possesses  an  almost  specific  action  for  fertilization 
of  the  sea  urchin  egg,  and  it  is  important  to  note  that  it 
increases  sperm  agglutination  also,  according  to  the 
same  author. 

We  shall  not  inquire  here  just  how  the  CaCl^  or 
NaOH  acts  in  such  cases,  but  will  hold  the  recorded 
facts  for  discussion  in  connection  with  other  data. 

VII.    OTHER   BLOCKS    TO   FERTILIZATION 

Any  environmental  defect  that  prevents  fertilization 
may  be  considered  as  a  block,  and  in  that  sense  the 
present  section  is  a  continuation  of  the  preceding.     It 


THE  PHYSIOLOGY  OF  FER 1 ILIZATION  1 73 

is  obvious  that  the  examination  of  conditions  that 
impede  or  prevent  fertilization  without  injury  to  the 
life  of  the  gametes  must  furnish  means  for  analysis. 

It  is  a  fact  well  known  to  embryologists  that  con- 
tamination of  the  eggs  of  some  marine  invertcl^rates 
with  blood  or  tissue  exudates  of  the  species  reduces 
considerably  the  percentage  of  fertilization;  it  is  there- 
fore a  common  practice  to  wash  the  eggs  once  or  several 
times  in  sea-water  before  insemination.  That  this 
effect  is  more  or  less  specific  was  proved  by  the  writer 
in  a  series  of  experiments,  hitherto  unpublished,  which 
show  that  the  filtered  plasma  of  the  coelomic  fluid  of 
sea  urchins  which  inhibits  fertilization  in  sea  urchins 
actually  increases  the  percentage  of  fertiHzation  in  the 
starfish. 

The  effect  in  the  case  of  the  sea  urchin  may  be 
shown  by  the  following  table: 

Percentage  of  _ 

Coelomic  Plasma  Percentage  of 

in  Sea-Water  Eggs  Segmented 

1 75 

5 10 

10 0.2 

20 0.2 

40 0.2 

100 o 

Control:  same  eggs  in  sea-water 75 

A  series  of  dilutions  of  the  filtered  plasma  was  made 
as  shown  in  the  left-hand  column;  and  identical  fcrtiU- 
zations  of  eggs  of  Arhacia  were  made  simultaneously  in 
each.  An  excess  of  sperm  was  used  in  each  case.  It 
is  clear  that  the  plasma  in  this  case  had  a  strong  inhib- 
iting effect  on  the  process  of  fertilization.  'The  inhibi- 
tion operates  on  the  initial  stages  because  membranes 


174  PROBLEMS  OF  FERTILIZATION 

are  not  formed  and  the  spermatozoa  do  not  penetrate 
into  the  egg,  ahhough  they  are  numerous  and  active. 
Other  samples  of  plasma  did  not  inhibit  so  strongly,  and 
very  wide  individual  variation  with  reference  to  this  effect 
was  found,  which  is  possibly  due  to  variation  in  the 
state  of  maturity  of  the  gonads.  The  same  principle 
holds  also  for  the  starfish;  thus  in  a  series  of  dilutions 
of  the  coelomic  fluid  of  the  starfish,  lo  per  cent,  20 
per  cent,  40  per  cent,  80  per  cent,  100  per  cent,  the 
percentages  of  segmentation  following  fertilization  of 
the  eggs  of  the  same  species  were  62  per  cent,  43  per 
cent,  50  per  cent,  32  per  cent,  26  per  cent,  11  per  cent; 
in  sea-water  98  per  cent  of  the  control  eggs  segmented. 
If  one  takes  Asterias  eggs  which  are  resistant  to 
fertiHzation  in  sea-water,  a  condition  often  found,  the 
addition  of  Arbacia  coelomic  fluid  may  prove  very  bene- 
ficial, as  the  following  experiment  shows:  a  series  of 
six  watch  glasses  was  laid  out  containing  5  c.c.  of  the 
following:  (i)  sea-water  (control),  (2)  5  per  cent  fil- 
tered plasma  of  Arbacia  in  sea-water,  (3)  10  per  cent  of 
the  same,  (4)  20  per  cent  of  the  same,  (5)  40  per  cent 
of  the  same,  (6)  80  per  cent  of  the  same.  Equal  quan- 
tities of  active  ^5/ma^  sperm  were  then  added  to  each, 
and  in  three  minutes  equal  quantities  of  Asterias  eggs 
which  had  stood  one  hour  in  sea-water  to  allow  them  to 
reach  the  most  favorable  stage  for  fertilization.  The 
percentages  of  eggs  that  segmented  were  (i)  o,  (2)  90 
per  cent,  (3)  90  per  cent,  (4)  40  per  cent,  (5)  10  per 
cent,  (6)  o.  In  (4),  (5),  and  (6)  membranes  formed 
though  the  eggs  did  not  segment.  This  experiment 
was  topical  of  many  performed.  The  reciprocal  experi- 
ment was  not  tried. 


THE  PHYSIOLOGY  OF  FERTILIZATION  175 

The  action  of  the  heterologous  plasma  may  be  sup- 
posed to  be  due  to  a  definite  membrane  effect  enabling 
interaction  between  sperm  and  egg  substances.  Its 
interest  in  the  present  connection  is  to  show  that  the 
inhibiting  effect  of  the  species  plasma  cannot  be  due 
merely  to  colloid  content,  which  is  common  to  both 
kinds  of  plasma,  but  that  it  has  something  specific 
in  it. 

Does  the  inhibitor  act  on  the  egg  or  on  the  sperm 
or  by  intervening  in  the  reaction  between  the  two  ? 
Experiments  undertaken  to  answer  this  question  showed 
that  both  eggs  and  sperm  exposed  to  the  plasma  and 
washed  free  of  it  again  were  fertilizable.  The  plasma 
therefore  merely  interferes  in  some  way  with  the  reac- 
tion. The  great  variability  of  the  effect  of  different 
samples  of  plasma  ranging  all  the  way  from  o  to  100  per 
cent  also  shows  that  we  are  not  deahng  merely  with 
a  general  colloid  effect. 

It  cannot  be  supposed  that  the  plasma  operates  by 
preventing  the  adhesion  of  the  spermatozoon  to  the  egg 
if  this  is  brought  about  by  agglutination,  because  it 
was  found  that  the  agglutination  of  spermatozoa  by 
means  of  egg  secretions  takes  place  as  readily  in  the 
plasma  as  in  sea-water. 

There  are  two  other  possibilities:  (i)  the  plasma 
might  be  supposed  to  harden  the  membrane  or  change 
its  permeable  character;  or  (2)  it  might  be  supposed 
to  inhibit  the  action  of  the  activable  substance  (ferti- 
lizin)  of  the  egg  by  deviation  effect.  If  the  latter  were 
true  it  should  then  be  possible  to  prevent  the  inhibitory 
action  of  the  plasma  by  first  saturating  it  with  the 
activable  substance.     This  was  found  to  be  true.     If  a 


176  PROBLEMS  OF  FERTILIZATION 

sample  of  plasma  be  divided  in  two  parts,  and  one  part 
be  saturated  with  egg  secretions  by  adding  a  large 
quantity  of  eggs,  it  is  found  that  this  portion  has  en- 
tirely lost  its  inhibiting  properties.  This  matter  will 
be  discussed  in  more  detail  in  a  later  section  (see 
chap.  vii). 

A  curious  form  of  inhibition  was  discovered  by 
Godlewski  (191 1)  and  described  by  him  under  the  name 
'^antagonism  of  sperm."  The  matter  was  subsequently 
investigated  by  Herlant  (191 2).  Godlewski  found  that 
sperm  of  the  annehd  Chaetopterus  would  call  forth  mem- 
brane formation  in  the  eggs  of  the  sea  urchin  Sphaer- 
echinus.  If,  however,  the  sperm  of  Chaetopterus  was 
mixed  in  proportions  ranging  from  equal  parts  to  two 
to  eight  with  the  sperm  of  S phaerechinus  no  egg  of 
Sphaerechinus  would  fertilize  in  the  mixture  after  it 
had  stood  a  few  minutes.  The  effect  developed  grad- 
ually. The  spermatozoa  were  perfectly  motile,  but  in 
some  way  they  antagonized  each  other's  action.  He  also 
found  that  the  sperm  of  Dentalium  (mollusk)  inhibited 
fertilization  of  sea  urchin  eggs  by  sperm  of  their  own 
species  used  in  the  same  way.  The  blood  also  of 
Chaetopterus  and  Dentalium  acts  in  the  same  way.  The 
eggs,  however,  remain  capable  of  fertilization  after  some 
time  in  the  mixture  by  fresh  species  sperm. 

This  author  therefore  attributed  the  effect  to  a  recip- 
rocal action  of  the  sperm  on  one  another  and  not  to 
an  action  of  the  mixture  on  the  eggs.  He  compared 
the  phenomenon  to  the  neutralizing  effect  which  dif- 
ferent cytolytic  sera  sometimes  exert  on  one  another, 
and  believed  that  it  probably  belonged  in  the  same 
category;   he  therefore  regarded  the  result  as  evidence 


THE  PHYSIOLOGY  OF  FERTILIZATION  177 

in  favor  of  Loeb's  hypothesis  that  the  fertiUzing  sul)- 
stance  of  the  spermatozoon  is  a  lysin. 

Herlant  subsequently  studied  the  same  phenomenon 
and  pointed  out  that  mere  dilution  of  the  sperm  mix- 
ture in  the  presence  of  eggs  with  pure  sea-water  would 
result  in  some  fertilizations,  and  that  eggs  that  had 
remained  even  seventy-five  minutes  in  the  mixture 
could  be  fertilized  with  fresh  species  sperm  if  they 
were  repeatedly  washed.  He  therefore  doubted  that 
there  was  any  profound  alteration  of  either  the  male 
or  the  female  sexual  elements,  and  postulated  somewhat 
doubtfully  a  physical  alteration  of  the  surface  of  the 
egg  in  the  presence  of  the  sperm  mixture. 

The  two  authors  thus  arrive  at  somewhat  different 
conclusions;  the  true  solution  may  involve  some  com- 
bination of  these  views.  The  present  writer  feels,  for 
reasons  discussed  later,  that  the  serum  analogy  of 
Godlewski  is  a  true  one  in  a  very  general  sense,  not 
that  the  sperm  carries  a  lysin,  a  conception  that  has  no 
longer  any  basis,  but  in  the  sense  that  reactions  com- 
parable to  serum  reactions  probably  are  involved  in 
fertilization.  If  we  were  to  suppose  that  the  foreign 
sperm  prevents  the  agglutination  of  the  species  sperm 
to  the  egg  we  would  perhaps  have  a  workable  hy- 
pothesis. 

In  the  cases  of  blood  inhibition  and  sperm  antago- 
nism we  have  two  forms  of  fertilization  blocks  that 
suggest  biochemical  factors  in  which  complex  substances 
play  a  role,  and  which  therefore  appear  to  belong  to  a 
different  category  from  deficiency  of  electrolytes  or 
acidity  effect. 


178  •    PROBLEMS  OF  FERTILIZATION 

REFERENCES 

BOVERI,   T. 

1889.  '*Ein  geschlechtlich  erzeugter  Organismus  ohne 
miitterliche  Eigenschaften,"  Sitzungsber.  d.  Gesell.  Jiir 
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Bryce  a:n»  Teacher. 

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Chambers,  Robert,  Jr. 

191 7.  "Microdissection  Studies.  II.  The  Cell  Aster:  a 
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XXIII,  483-504. 

CoHN,  Edwin  J. 

1918.  See  references  at  end  of  chapter  iv. 

Delage,  Y. 

1898.     "Embryons  sans  noyau  maternal,"  Comptes  rendus 

de  VAcad.  des  Sci.,  T.  127,  pp.  528-31. 
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gen.,  Ser.  3,  T.  7,  pp.  383-417. 
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et  sur  une  theorie  nouvelle  de  la  fecondation  nor- 

male,"  ibid.,  pp.  512-27. 
1901a.     "Etudes  experimentales  sur  la  maturation  cytolo- 

gique   et   sur   la  parthenogenese  artificielle  chez  les 

echinodermes,"  ibid.,  T.  9,  pp.  285-326. 
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piires  et  appL,  T.  12,  pp.  864-74. 

DuNGAY,  Neil  S. 

1 913.  "A  Study  of  the  Effects  of  Injury  upon  the  Ferti- 
lizing Power  of  Sperm,"  Biol.  Bull.,  XXV,   213-60. 

FucHS,  H.  M. 

1914.  "The  Action  of  Egg-Secretions  on  the  Fertilizing 
Power  of  Sperm,"  Arch.  Jiir  Entwickelungsmech., 
Band  42,  pp.  205-52. 


THE  PHYSIOLOGY  OF  FERTILIZATION  179 

FucHS,  H.  M. 

1915-  "Studies  in  the  Physiology  of  Fertilization,"  Jour, 
of  Genetics,  IV,  259-301. 

Gemmill,  James  J. 

1900.  See  references  at  end  of  chapter  iv. 
GiES,  W.  J. 

1901.  "Do  Spermatozoa  Contain  an  Enzyme  Having  the 
Power  of  Causing  the  Development  of  Mature  Ova  ?  " 
Am.  Jour,  of  Physiol.,  VI,  53. 

Glaser,  Otto. 

1914a.     See  references  at  end  of  chapter  iv. 
•    1914/7.     "The  Change  in  Volume  of  Arbacia  and  Aslerias 
Eggs  at  Fertilization,"  Biol.  Bull.,  XXVI,  84-91. 

GODLEWSKI,    EmIL. 

191 1.  "Studien  iiber  die  Entwicklungserregung:  II,  Antag- 
onismus  der  Einwirkung  des  Spermas  von  verschic- 
denen  Tierklassen,"  Arch,  fiir  Entwickelungsmech., 
Band  ^2>y  PP-  233-54- 

191 2.  "Studien  iiber  die  Entwicklungserregung,  ibid.,  pp. 
196-254. 

Gray,  J. 

1 9 13.  "The  Electrical  Conductivity  of  Fertilized  and  Un- 
fertilized Eggs,"  Jour.  Marine  Biol.  Assoc,  pp.  50-59. 

Harvey,  E.  N. 

1910.  "The  Permeability  and  Cytolysis  of  Eggs,"  Science, 
N.S.,  XXXII,  565-68. 

Heilbrunn,  L.  V. 

1915.  "Studies  in  Artificial  Parthenogenesis.  II.  Physical 
Changes  in  the  Egg  of  Arbacia,"  Biol.  Bull.,  XXIX, 
149-203. 

Herbst,  Curt. 

1909.  "Verebungstudien.  VI,"  Arch,  fiir  Enhcichelungs- 
mech..  Band  27,  pp.  266-308.  See  also  ibid.  (191 2), 
Band  34,  pp.  1-89. 

Herlant,  Maurice. 

191 2.  "Recherches  sur  I'antagonisme  dc  deux  spcrmes 
provenant  d'especes  cloignees,"  Anal.  Anz.,  Band 
42,  pp.  563-75- 


i8o  PROBLEMS  OF  FERTILIZATION 

Hertwig,  O.  and  R. 

1887.     See  references  at  end  of  chapter  i. 

191 1.     Die  Radiumkrankheit  tierischer  Keimzellen;  ein  Bei- 

trag  zur  experimenteller  Zeugungs-  und  Vererbungs- 

lehre.     Bonn:   Friedrich  Cohen. 

Just,  E.  E. 

1915a.    "An  Experimental  Analysis  of  Fertilization  in  Pla- 

tynereis  megalops,"  Biol.  Bull.,  XXVUI,  93-114. 
191 5^.    "Initiation  of  Development  in  Nereis,  ibid.,  pp.  1-17. 
1 91 9.     See  references  at  end  of  chapter  iv. 

LiLLiE,  Frank  R. 

191 1.  "Studies  of  Fertilization  in  Nereis.  II.  Partial  Ferti- 
lization," Jour,  of  M  or  ph.,  XXII. 

191 2.  "Studies  of  Fertilization  in  Nereis.  IV.  The  Ferti- 
lizing Power  of  Portions  of  the  Spermatozoon,"  Jour. 
Exp.  Zool.,  XII,  427-76. 

19 13.  See  references  at  end  of  chapter  iv. 

1914.  See  references  at  end  of  chapter  iv. 

191 5.  See  references  at  end  of  chapter  iv. 

LiLLIE,   R.   S. 

191 5.  "On  the  Conditions  of  Activation  of  Unfertilized 
Starfish  Eggs  under  the  Influence  of  High  Tempera- 
tures and  Fatty  Acid  Solution,"  Biol.  BiilL,  XXVIII, 
260-303. 

1916.  "Increase  of  Permeability  to  Water  Following  Nor- 
mal and  Artificial  Activation  in  Sea-Urchin  Eggs," 
Am.  Jour,  of  Physiol.,  XL,  249-66. 

191 7.  "The  Conditions  Determining  the  Rate  of  Entrance 
of  Water  into  Fertilized  and  Unfertilized  Arhacia 
Eggs,  and  the  General  Relation  of  Changes  of  Per- 
meability to  Activation,"  ibid.,  XLIII,  43-57. 

1918.  "The  Increase  of  Permeabihty  to  Water  in  Ferti- 
lized Sea-Urchin  Eggs  and  the  Influence  of  Cyanide 
and  Anaesthetics  upon  this  Change,"  ibid.,  XLV, 
406-30. 

LoEB,  Jacques. 

1908.  "Ueber  die  osmotischen  Eigenschaften  und  die  Ent- 
stehung  der  Befruchtungsmembran  beim  Seeigelei," 
Arch,  fiir  Entwickelungsmech.,  Band  26,  pp.  82-88. 


THE  PHYSIOLOGY  OF  FERTILIZATION  i8r 

LoEB,  Jacques. 

1913a.   Artificial  Parthenogenesis  and  Fertilization.    Chicago- 

I  he  University  of  Chicago  Press  ' 

1913b.    ''Reversibility   in    Artificial    Parthenogenesis,"   Sd- 

ence,  N.S.,  XXXVIII,  749-50 

'  '914.     ''On  Some  Non-specific  Factors  for  the  Entrance  of 

the^Spermatozoon  into  the  Egg,"  Science,  N.S.,  XL, 

1915a.  ''Reversible  Activation  and  Incomplete  Membrane 
Formation  of  the  Unfertilized  Eggs  of  the  Sea- 
Urchin,"  Biol.  Bull.,  XXIX,  103-10 
--1915b.  "On  the  Nature  of  the  Conditions  Which  Determine 
or  Prevent  the  Entrance  of  the  Spermatozoon  into 
the  Egg,    Am.  Naturalist,  XLIX,  257-85. 

LOEB,   J.,    AND   WaSTENYS,    H. 

1912.     ''DieOxydationsvorgange  im  befruchteten  und  unbe- 
fruchteten  Seesternei,"  Arck.  fiir  Entwickelungsmcch 
Band  35,  pp.  555-57. 

1913^.  "The  Relative  Influence  of  Weak  and  Strong  Bases 
upon  the  Rate  of  Oxydations  in  the  Unfertilized  Egg  of 
the  Sea-Urchin,"  Jour,  of  Biol.  Chem.,  XIV,  355-61 

1913^'.  ''The  Influence  of  Bases  upon  the  Rate  of  Oxida- 
tions in  Fertilized  Eggs,"  ibid.,  459-64. 

1913^.  "The  Influence  of  Hypertonic  Solution  upon  the 
Rate  of  Oxydations  in  Fertilized  and  Unfertili/cd 
Eggs,"  ibid.,  469-80. 

1915.     ''Further    Experiments    on    the    Relative    EflVct    of 
Weak  and  Strong  Bases  on  the  Rate  of  Oxydations 
in  the  Egg  of  the  Sea-Urchin,"  ibid.,  XXI   ic^i-cS 
Lyon,  E.  P.  -  ^    :>:>:>  - 

1909.  "The  Catalaze  of  Echinoderm  Eggs  before  and  after 
Fertilization,"  Am.  Jour,  of  Physiol.,  XX\',  199-213. 

Lyon,  E.  P.,  and  Shackell,  L.  F. 

1910.  "On  the  Increased  Permeability  of  Sea-Urchin  Eggs 
Following    Fertilization,"    Science,     N.S.,     XXXU 
249-51. 

IMcClendon,  J.  J. 

1910.  "Electrolytic  Experiments  Showing  Increase  of 
Permeabihty  of  the  Egg  to  Ions  at  the  Beginning  of 
Development,"  ^-r/V^rf,  N.S.,  XXXn,  1 22-24,  3  r  7-18 


1 82  PROBLEMS  OF  FERTILIZATION 

Mall,  F.  P. 

1918.  *'0n  the  Age  of  Human  Embryos,"  Am.  Jour,  of 
Anat.,  XXIII,  397-422. 

Moore,  Carl. 

1916.  ''On  the  Superposition  of  FertiHzation  and  Parthe- 
nogenesis," Biol.  Bull.,  XXXI,  137-80. 

191 7.  "On  the  Capacity  for  Fertilization  after  the  Initia- 
tion of  Development,"  ibid.,  XXXIII,  258-95. 

Morgan,  T.  H. 

1895.  "The  Fertilization  of  Non-nucleated  Fragments  of 
Sea-Urchin  Eggs,"  Arch,  fiir  Entwickelungsmech., 
Band  2,  pp.  268-80. 

Okkelberg,  Peter. 

1914.  "Volumetric  Changes  in  the  Egg  of  the  Brook  Lam- 
prey, Entosphenus  (Lampetra)  Wilderi  (Gage),  after 
Fertilization,"  Biol.  Bidl.,  XX\T,  92-99. 

Packard,  Charles. 

1914.  "The  Effect  of  Radium  Radiation  on  the  Fertiliza- 
tion of  Nereis,''  Jour.  Exp.  Zool.,  XVI,  85-131. 

Reighard,  J.  E. 

1893.  "The  Ripe  Eggs  and  the  Spermatozoa  of  the  Wall- 
eyed Pike  and  Their  History  until  Segmentation 
Begins,"  Tenth  Bienn.  Kept.  State  Board  of  Fish 
Comm.  of  Mich.    Lansing. 

Robertson,  T.  Brailsford. 

1912a.  "On  the  Extraction  of  a  Substance  from  the  Sperm 
of  a  Sea  Urchin  {Strongylocentrotiis  purpuratus)  Which 
Will  Fertilize  the  Eggs  of  that  Species,"  Jour,  of 
Biol.  Chem.,  XII,  i-ii. 

191 2/).  "Studies  in  the  Fertilization  of  the  Eggs  of  a  Sea- 
Urchin  (Strongylocentrotus)  by  Blood-Sera,  Sperm, 
Sperm-Extract,  and  Other  FertiHzing  Agents,"  Arch, 
fiir.  Entwickelungsmech.,  Band  35,  pp.  64-130. 

SCHUCKING,   A. 

1903.     See  references  at  end  of  chapter  iv. 


THE  PHYSIOLOGY  OF  FERTHJZATION  183 

Seeliger,  O. 

1894.  ''Giebt  es  geschlechtlich  erzeugle  Organismcn  ohne 
mutterhche  Ligenschaften  ?"  Arch,  fur  EutunckclunZ 
mech.,  Band  i,  p.  203.  ^ 

^^96.  '/Bemerkungen  liber  Baslardlarven  der  Sceigel  " 
tbtd.,  Band  3,  pp.  477-526.  ^    ' 

Stockard,  Charles  R.,  and  Craig,  Dorothy. 

1904.     ''An  Experimental  Study  of  the  Influence  of  Alcohol 
on  the  Germ-Cells  and  the  Developing  Embrx'o  of 
Mammals,"  Arch,  fur  Entmckelungsmech.,  Band  ,4 
pp.  569-84.  ^^' 

Triepel,  a. 

1914,  1915.     "Alterbestimmung  bei  menschlichen  Embr^'o- 
nen,    Anal.  Anz.,  Band  46,  Band  48. 
Warburg,  Otto. 

1908.  "Beobachtungen    Uber    die    Oxydationsprocesse    im 
Seeigele.,"  ZeUschr.  jur  physiol.  Chem.,  Band  .,7    pn 

I-I6.  •^''    ^^' 

1909.  -Ueber  die  Oxydationen  im  Ei:  II,  Mittheilung," 
^bld.,  Band  60,  pp.  443-52. 

1910.  -Ueber  die  Oxydationen  in  lebenden  Zellen  nach 
Versuchen  am  Seeigelei,"  ibid.,  Band  66,  pp.  305-40 

1914..  ''Ueber  die  Rolle  des  Eisens  in  der  Atmung  des 
Seeigeleies  nebst  Bemerkungen  uber  einige  durch 
Eisen  beschleunigte  Oxydationen,"  ibid,  Band  92,  pp. 

1914/^.     ''Zellstruktur  und  Oxydationsgeschwindigkeit  nach 
Versuchen  am  Seeigelei,"  PflUgcr^s  Arch.  Jiir  die  ges. 
Physiol.,  Band  158,  pp.  189-208. 
Wilson,  E.  B. 

1903.     ''Experiments  on  Cleavage  and  Localization  in  the 
Nemertean  Egg,"  Arch,  fur  Enlwickelmigsmcch.,  Band 
16,  pp.  411-60. 
Winkler,  H. 

1900.     "Ueber  die  Furchung  unbefruchteter  Eier  unter  der 
Emwirkung  von  Extractivstoffen  aus  den  Sperma  " 
Nachrichlen  d.  Gesell.  d.  Wiss.     Gollingcn 
Ziegler,  H.  E. 

1898.     "Experimentelle    Studien    iiber    die    Zelltheilung " 
Arch,  far  Enlmickcliuigsmech.,  Band  4,  pp.  249-93. 


CHAPTER  VI 
THE  PROBLEM   OF   SPECIFICITY  IN  FERTILIZATION 

In  fertilization  we  have  both  tissue  and  species 
specificity.  The  spermatozoon  does  not  react  with, 
nor  penetrate,  other  kinds  of  cells  than  the  ovum 
(tissue  specificity),  a  fact  that  suggests  the  question 
whether  this  is  due  to  a  chemical  specificity  of  the  egg 
or  merely  to  its  physical  characteristics.  Fertilization 
is  also  species  specific,  but  only  in  a  restricted  sense, 
because  it  is  possible  to  a  certain  extent  between  dif- 
ferent species  or  even  wider  groups  (hybridization); 
the  degree  of  this  specificity  is  subject  to  rather  wide 
variations  in  different  animal  groups,  but  it  is  in  a 
very  general  sense  defined  by  taxonomic  relations. 
Within  the  species  also  there  are  undoubtedly  individ- 
ual variations  in  fertility,  which  reach  an  extreme  in 
the  phenomenon  of  self -sterility  in  certain  ascidians. 

I.    TISSUE    SPECIFICITY 

Kohlbrugge  (1910,  191 1,  191 2)  maintains  that  sper- 
matozoa freely  penetrate  the  epithelial  cells  of  the 
uterine  mucosa  in  animals  with  internal  fertilization 
that  he  has  studied  (bat,  mouse,  rabbit,  hen,  dogfish, 
skate)  and  may  even  pass  through  these  into  the  under- 
lying connective  tissue.  Sobotta  (191 1)  was  unable  to 
confirm  the  observations  (on  the  mouse)  and  showed 
that  most  of  the  spermatozoa  degenerate  without  free- 
ing themselves  from  the  ejaculate.  He  criticizes  Kohl- 
brugge severely  for  his  methods,  and  suggests  that  the 

184 


SPECIFICITY  IN  FER  riLIZATIOX  185 

bodies  identified  by  the  latter  as  sperm  heads  in  the 
uterine  epithelium  may  be  shrunken  nuclei.  Rohl- 
brugge  has  gone  as  far  as  to  suggest  that  there  ma\' 
be  a  fusion  of  the  sperm  nuclei  and  the  nuclei  of  epi- 
thelial cells  and  a  consequent  stimulation  of  cell  division 
and  growth;  but  the  evidence  offered  for  these  sugges- 
tions is  highly  unsatisfactory.  However,  he  presents 
his  observations  as  a  possible  explanation  of  telegony, 
a  phenomenon  which  has  no  present  status  among 
biologists.  The  observations  of  Kohlbrugge  require 
confirmation  before  they  can  be  accepted  as  a  body 
of  ascertained  fact.  The  same  author  has  also  main- 
tained that  spermatozoa  may  enter  cleavage  cells,  or 
cells  of  the  blastodermic  vesicle  of  mammals;  but  here 
also  confirmation  is  lacking. 

There  is  some  evidence  from  the  clinical  side  that 
the  sperm  of  the  male  influences  the  body  of  the  female, 
and  Waldstein  and  Eckler  (1913)  niaintain  that  a 
specific  ferment  develops  in  the  blood  of  rabbits  a  few 
hours  after  coitus  which  is  directed  against  sperm,  and 
may  be  detected  by  the  method  of  Abderhalden.  This 
would  demonstrate  absorption  of  sperm,  but  not  neces- 
sarily in  the  manner  described  by  Kohlbrugge. 

In  a  considerable  number  of  animals  spermatozoa 
find  their  way  among  the  tissues  either  as  a  result  of 
the  method  of  copulation  or  otherwise.  But  they  arc 
not  known  to  react  with  any  other  cells  than  the  ova. 
There  is  quite  an  extensive  literature  on  this  subject ; 
the  principal  references  are  Whitman  (1891),  Kohl- 
brugge (1910,  191 1,  191 2),  Berlese  (1898),  etc. 

The  problem  of  tissue  specificity  as  between  ovum 
and  spermatozoa   has    not,    however,   attracted    much 


1 86  PROBLEMS  OF  FERTILIZATION 

attention.  The  failure  of  the  spermatozoa  to  enter 
other  kinds  of  cells,  even  though  there  is  abundant 
opportunity  for  it,  requires  some  explanation.  In  the 
fertilization  of  Ascaris  megalocephala,  for  instance,  the 
spermatozoa  find  their  way  up  the  long  oviduct  and 
may  be  found  in  considerable  numbers  in  the  interstices 
between  the  enormous  swollen  epithelial  cells  of  the 
oviduct,  but  they  never  enter  the  latter;  in  the  lumen 
of  the  oviduct  the  membraneless  eggs,  hardly  larger  than 
the  oviducal  cells,  are,  however,  all  fertilized.  The  recip- 
rocal of  this  relation  is  found  in  sperm  agglutination 
by  secretions  of  ova  but  by  no  other  tissue  products. 
The  writer  (1913)  studied  the  latter  question  in  detail 
in  the  sea  urchin  and  in  Nereis  and  was  unable  to  derive 
even  a  trace  of  sperm-agglutinating  substance,  either 
from  the  blood,  which  must  contain  secretions  of  all 
tissues,  or  from  any  of  the  tissues  individually;  but 
the  ripe  eggs  always  produced  the  agglutinating  sub- 
stance abundantly.  There  is  thus  a  definite  tissue 
specificity  in  fertilization  due  to  specific  chemical 
organization  of  the  gametes,  as  one  factor  at  least. 

II.       SPECIES    SPECIFICITY 

I.  Hybrid  fertilization. — The  extent  to  which  species 
specificity  in  fertiHzation  is  due  to  actual  differences  in 
the  chemical  make-up  of  interacting  substances  has 
caused  much  discussion.  The  problems  united  under 
this  head  ally  themselves  with  problems  of  tissue  and 
blood  specificity  and  have  the  broadest  biological  bear- 
ing. Specificity  may  concern  different  stages  of  ferti- 
lization; in  certain  hybrid  combinations  the  eggs  do 
not  appear  to  react  at  all  to  the  foreign   sperm;    in 


SPECIFICITY  IN  FERTILIZATIOX  187 

others  the  foreign  sperm  may  produce  cortical  changes 
but  fail  to  penetrate;  or  the  sperm  may  penetrate,  and 
perish  without  uniting  with  the  egg  nucleus;   or  it  may 
unite  and  be  secondarily  eliminated  in  the  first  or  later 
cleavages;   or  again,  without  elimination,  the  combina- 
tion may  prove  its  incompatibility   by  abnormalities 
m  development  appearing  rapidly  or  slowly;    in   yet 
other  cases  the  hybrid  may  develop  fully  but  remain 
sterile.     Finally  we  may  have  completely  fertile  hybrids 
from   certain   crosses   of   closely  related   species.     The 
blocks  to  hybrid  fertilization  are  thus  not  the  same  in 
all  cases;    even  in  the  case  of  cortical  block  it  is  quite 
conceivable  that  we  have  different  causes  operative  in 
different  cases.     This  block  seems  to  be  readily  remo\- 
able  in  some   instances  by  mere  increase  in  alkalinity 
of    the   medium  as  first  shown   by  Loeb,  but   this   is 
by  no  means  always  so.     The  internal  blocks  on   the 
other  hand  are  not  controllable  by  means  hitherto  em- 
ployed. 

From  the  standpoint  of  preservation  of  the  species 
it  makes  but  little  difference  in  what  stage  hybrid  fer- 
tilization exhibits  its  incompatability  so  long  as  the 
hybrid  does  not  breed.  But  from  the  standpoint  of 
fertilization  problems  we  need  consider  only  the  incom- 
patibilities of  the  stages  of  fertilization  itself. 

A  brief  systematic  survey  of  the  field  will  prepare 
the  way  for  consideration  of  the  problems.  The 
groups  in  which  the  possibilities  of  hybrid  fertilization 
have  been  most  fully  investigated  are  the  echinoderms, 
teleosts,  and  amphibia.  In  some  groups,  as  in  insects, 
birds,  and  mammals,  mating  behavior  constitutes  a 
serious  obstacle  to  close  investigation. 


1 88  PROBLEMS  OF  FERTILIZATION 

a)  Echinoderms:  We  may  consider  the  data  in 
taxonomic  order:  (i)  species  crosses,  (2)  genus,  fam- 
ily, and  ordinal  crosses,  (3)  class  crosses,  (4)  phylum 
crosses. 

I.  Shearer,  De  Morgan,  and  Fuchs  (1913)  crossed 
three  species  of  Echinus:  esculentus,  acutus,  and  mili- 
aris,  in  all  possible  combinations.  The  fertilization  suc- 
ceeded in  all  six  combinations  without  any  artificial  aid, 
by  either  increasing  the  usual  concentration  of  the 
sperm  or  changing  the  chemical  composition  of  the 
sea-water.  Larvae  were  readily  raised  from  all  crosses, 
but  only  the  crosses  E.  esculentus  ?  X  £.  acutus  6 
and  E.  miliaris  ?  X  £.  acutus  male  gave  normal  sea 
urchins. 

The  cytology  of  these  crosses  was  studied  by  Don- 
caster  and  Gray  (1913)  and  by  Gray  (1913).  The  be- 
havior of  the  germ  nuclei  was  normal;  but  some  elim- 
ination of  chromosomes  from  the  first  cleavage  spindle 
occurred  in  certain  of  the  crosses.  A  curious  fact  was 
that  this  might  occur  in  one  reciprocal  of  a  cross  but 
not  in  the  other.  Thus  in  the  cross  esculentus  ?  X 
acutus  6  the  cytological  events  are  perfectly  normal; 
but  in  acutus  9  X  esculentus  6  there  was  an  invariable 
elimination  of  some  chromosomes.  In  this  process  vesi- 
cles formed  on  certain  chromosomes,  and  often  sepa- 
rated from  them  and  came  to  lie  outside  of  the  spindle; 
other  altered  chromosomes  were  often  carried  entire  to 
one  pole  without  dividing.  It  was  not  possible  to  deter- 
mine what  relation  there  might  be  between  chromosome 
elimination  and  the  character  of  inheritance. 

Such  elimination  of  chromosomes  from  hybrid  zy- 
gotes has  often  been  referred  vaguely  to  incompatibility; 


SPECIFICITY  IN  FERTILIZATION  189 

but  in  this  case  it  is  difficult  to  suggest  why  it  should 
exist  in  one  reciprocal  and  not  in  the  other.  Gray 
(1913)  found  that  treatment  of  fertilized  eggs  of  E. 
acutus'^iXh  hypertonic  solutions  of  medium  strength 
caused  elimination  of  some  chromosomes,  but  the 
phenomenon  could  not  be  induced  in  E.  esculenlus  by 
similar  treatment.  He  suggests  that  the  chromosome 
behavior  in  the  reciprocal  crosses  might  be  understood 
on  the  assumption  that  the  osmotic  relations  are  different 
in  the  cross  and  pure  species,  owing  to  different  effects, 
on  permeability  of  the  egg,  of  the  foreign  and  species 
sperm.  This  would  imply  that  the  eliminated  chromo- 
somes are  of  maternal  origin;  however,  this  cannot  be 
proved  in  the  cross  under  consideration,  and  it  is  known 
that  in  wider  crosses  the  eliminated  chromosomes  are 
of  paternal  origin  generally  (Herbst,  Balzer,  Tenncnt). 

2.  Interspecific  crosses  among  the  echinoderms  seem 
to  have  been  confined  to  the  genus  Ecliinus,  and  we 
are  therefore  unable  to  make  any  general  statement 
concerning  the  possibilities.  But  wider  crosses  within 
the  order  have  been  made  very  frequently.  W^rnon 
(1900)  alone  attempted  forty-nine  out  of  a  possible 
fifty-six  cross-fertilizations  between  eight  species  belong- 
ing to  seven  genera  of  sea  urchins;  only  eleven  of  these 
gave  no  sign  of  cross-fertilization;  of  the  remainder, 
nine  gave  only  segmentation  stages  or  blastulae  or 
gastrulae,  and  twenty-nine  lived  to  the  stage  of  eight - 
day  plutei. 

In  Vernon's  cross-fertilizations  a  high  sperm  con- 
centration seems  generally  to  have  been  employed. 
The  percentage  of  eggs  fertilized  was  nevertheless  small 
as  compared  with  species  fertilization;    in  many  cases 


IQO  PROBLEMS  OF  FERTILIZATION 

exceedingly  small.  In  several  instances  the  eggs  were 
staled  for  several  hours,  even  up  to  twenty-four,  before 
.  fertilization,  with  resulting  increase  in  the  percentage  of 
eggs  fertihzed;  but  in  the  case  of  S phaerechinus  ferti- 
lized by  Strongylocentrotus  this  treatment  was  not  suc- 
cessful. It  is  important  to  note  that  specificity  always 
appeared  with  reference  to  the  relative  ease  of  fertiliza- 
tion with  the  specific  and  foreign  sperm. 

Tennent  (1910)  made  eleven  crosses  within  the  order 
in  which  the  reciprocals  belonged  to  different  genera, 
famihes,  or  suborders;  and  many  cross-fertilizations 
within  the  order  have  been  made  by  other  observers. 
Unfortunately  for  our  purpose  these  studies  have  been 
made  from  the  point  of  view  of  heredity  or  chromo- 
some behavior,  and  the  fertilization  problems  have  been 
referred  to  only  incidentally  as  a  general  rule.  They 
are,  however,  sufficient  to  show  that  the  chances  of 
success  of  a  cross  cannot  be  postulated  wholly  on  the 
systematic  position  of  the  species.  Thus  Tennent 
reports  that  the  cross  between  Moira  ?  and  Toxo- 
pneustes  6  belonging  to  different  suborders  takes  place 
very  readily  and  the  larvae  develop  well.  The  recip- 
rocal cross  can  also  be  made,  but  succeeds  best  if  the 
Toxopneustes  eggs  are  allowed  to  stand  in  sea-water 
five  hours  before  being  fertilized.  Hipponoe  ?  crossed 
with  Cidaris  6,  also  belonging  to  different  suborders, 
gives  poor  results;  no  fertilization  membrane  is  formed, 
segmentation  is  irregular,  larvae  abnormal. 

Reciprocal  crosses  are  sometimes  quite  similar  with 
reference  to  fertilization;  but  frequently  they  are  not. 
Thus  Fischel  (1906)  reports  for  the  crosses  between 
Strongylocentrotus  and  Arhacia  that  fertilization  never 


SPECIFICITY  IN  FERTILIZATIOX 


191 


succeeds  when  Slrongylocentrotus  is  the  male,  but  always 
succeeds  when  Arbacia  is  the  male.  He  states  that, 
curiously  enough,  the  Hertwigs  obtained  the  exact  oppo- 
site result  with  these  genera  in  another  locality.  Some 
authors  state  only  their  successful  cross-fertilizations, 
and  the  facts  with  respect  to  the  reciprocals  are  not 
stated. 

Balzer  (1910)  has,  however,  paid  particular  atten- 
tion to  this  question,  and  his  results  may  be  tabulated 
as  follows: 


Echinus  ? 

Slrongylocentrotus  $ 

Sphaerechinus  ? 

1 

'             Arbacia  ? 

Echimis  $ 

No  elimiiiation 

(i) 

No  elimina- 
tion (2) 

No  elimina- 
tion (3) 

No  elimina- 
tion (6) 

28-30  chromosomes 
eliminated  first 
cleavage  (4) 

Slrongylo- 
centrotus 0 

No  elimination 

(i) 

28-30  chromosomes 
eliminated  first 
cleavage  (5) 

Sphaere- 
chinus  S 

21  chromosomes 
eliminated  first 
cleavage  (2) 

21  chromosomes 
eliminated  first 
cleavage  (3) 

21  chromosomes 
eliminated  first 
cleavage  (6) 

Arbacia  $ 

Chromatin  elim- 
inated blastula 
(4) 

Chromatin  elim- 
inated blastula 
(5) 

Note. — The  reciprocal  crosses  bear  the  same  number. 

« 

Only  three  of  these  cross-fertilizations  were  success- 
ful in  sea-water,  then  only  occasionally,  and  quanti- 
tative data  were  not  given,  viz.:  Sphaerechinus  v^  X 
Slrongylocentrotus  c?;  Slrongylocentrotus  ?  X  Arbacia  ^; 
Echinus  ?  X  Sphaerechinus  S.  The  fertilizations  re- 
corded were  therefore  made  in  hyperalkaline  sea-water 
according  to  Loeb's  method.  Very  great  variability  in 
the  capacity  for  hybridization  even  b\'  this  method  was 


192  PROBLEMS  OF  FERTILIZATION 

encountered  as  by  other  authors.  But  Httle  can  be 
learned,  therefore,  concerning  the  kind  and  degree  of 
the  natural  specificity  of  the  fertilization  reactions  in 
these  cases,  except  what  has  been  stated,  viz.,  that  in 
nine  out  of  the  twelve  cross-fertilizations  hyperalkahne 
conditions  are  necessary  to  permit  penetration  of  the 
spermatozoon.  But,  after  the  cortical  specificity  is 
broken  down  by  the  hyperalkahne  medium,  fertihza- 
tion  proceeds  normally  up  to  the  metaphase  of  the 
first  cleavage,  when  elimination  of  paternal  chromo- 
somes occurs  regularly  in  certain  cases.  In  other  cases 
ehmination  of  paternal  chromatin  is  postponed  until 
the  blastula  stage,  and  in  yet  others  it  does  not  occur 
at  all.  But  there  is  only  one  case  in  which  the  recip- 
rocals behave  alike  in  this  respect,  viz.,  in  the  Echi- 
nusXStrongylocentrotus  crosses,  among  the  six  pairs  of 
reciprocals. 

There  is  certainly  a  quantitative  specificity  in  these 
cases.  For  a  critical  examination  of  the  problem  of 
specificity  within  the  class  we  need  a  quantitative  con- 
trol of  egg  and  sperm  concentration  for  the  specific  and 
the  cross-fertilizations  which  should  be  reciprocal,  and  a 
careful  cytological  examination  of  tfie  eggs.  In  the 
second  place  we  need  also,  in  all  cases  in  which  the 
gametes  are  refractory  to  cross-fertilization,  an  exper- 
imental testing  of  methods  for  overcoming  the  diffi- 
culty. Until  this  is  done  we  cannot  say  what  the 
measure  of  specificity  really  is.  In  the  third  place  the 
viabihty  of  the  crosses  should  be  thoroughly  tested; 
it  has  been  shown  in  many  cases  that  development 
becomes  abnormal  in  the  blastula  or  gastrula  and,  in 
other  cases,  in  pluteus  stages.     But  some  crosses  even 


SPECIFICITY  IN  FERTILIZATIOX 


93 


between  suborders  give  strong  plutei;  however,  no  one 
has  carried  the  crosses  considered  in  this  section  through 
the  metamorphosis,  and  it  is  not  known  whether  this 
is  possible. 

The    heredity    of    intergeneric    and    wider    crosses 
within  the  order  is  a  matter  of  great  interest,  but  the 
discussion   would   carry   us   beyond    the   scope   of  our 
subject.     There  is  a  strong  tendency  for  such  crosses 
to    exhibit    a   preponderance    of    maternal    characters, 
though   this   is   not   always   the   case.     Herbst   (1909,' 
1912)  and  Balzer  (1910)  have  shown  that  such  cases 
are,  frequently  at  least,  accompanied  by  an  extrusion 
of  paternal  chromatin,  and  they  have  thus  furnished 
an  explanation  of  some  cases  of  matroclinal  heredity. 
But  other  authors  have  pointed  out  that  the  direction 
of  inheritance  may  be  related  to  reaction  of  the  sea- 
water  (Tennent),  or  to  seasonal  conditions  (Vernon), 
or   to  factors  affecting  the  growth  of   the  genyi  cells 
during  their  period  of  growth  and  maturation  (Shearer, 
De  Morgan,  and  Fuchs).     These  views  are  not  neces- 
sarily inconsistent,  for  the  external  factors  may  operate 
by  modification  of  chromosome  relations. 

3.  Interclass  crosses  have  also  been  made  in  echino- 
derms.  In  all  such  cases  it  has  been  necessary  to 
create  artificial  conditions  for  the  fertilization  reaction. 
The  first  experiments  of  this  kind  were  performed  b\- 
Loeb  in  1903,  who  found  that  the  eggs  of  Slroni^vlo- 
centrotus  purpuratiis  can  be  fertilized  by  the  sperm  of 
Asterias  (starfish)  in  the  presence  of  an  excess  of  alkah'. 
Fertilization  succeeded  best  in  a  solution  of  100  c.c. 
sea-water  to  which  1.2  c.c.  N/io  XaOH  had  bcrn 
added.     Perhaps  50  per  cent  formed  membranes  and 


194  PROBLEMS  OF  FERTILIZATION 

segmented,  and  many  lived  until  the  third  day  and 
formed  a  rudimentary  skeleton,  but  then  died.  The 
characters  developed  were  exclusively  maternal.  The 
sperm  of  certain  other  starfishes  was  less  effective,  but 
that  of  a  brittle  star  was  equal  to  Asterias.  Only  those 
eggs  developed  into  which  a  spermatozoon  penetrated, 
but  some  eggs  which  formed  membranes  failed  to  seg- 
ment, and  Loeb  and  his  students  showed  that  in  these 
cases  the  membrane  had  been  caused  by  external  action 
of  the  spermatozoon  alone.  Loeb  could  not  succeed 
in  fertilizing  the  eggs  of  Arbacia  with  the  sperm  of 
Asterias  by  this  method. 

The  starfish  sperm  affect  the  sea  urchin  egg  only  in 
the  presence  of  the  alkali;  eggs  or  sperm  previously 
treated  with  alkah  will  not  react  when  brought  together 
in  pure  sea-water.  The  effect  may  be  on  the  spermato- 
zoon or  on  the  egg  or  on  both;  but  it  is  obvious  that 
some  surface  reaction  of  the  egg  and  spermatozoon  is 
favored  by  the  alkali,  because  when  the  sperm  once 
gains  entrance  to  the  egg  it  calls  forth  the  further 
necessary  reactions  within  it. 

In  attempting  a  further  analysis  of  this  subject 
Loeb  (19 14)  discovered  that  if  the  sea  urchin  eggs  are 
deprived  of  their  jelly  by  action  of  HCl  they  cannot 
be  fertilized  by  starfish  spermatozoa  with  the  same  use 
of  hyperalkahne  sea-water  that  readily  brings  about 
fertilization  in  the  presence  of  the  jelly,  although  they 
are  readily  fertilized  with  their  own  sperm.  But  if  an 
excess  of  calcium  is  added  to  the  hyperalkahne  sea- 
water  the  heterogeneous  fertilization  succeeds  in  the 
absence  of  the  jelly,  and  in  this  case  practically  all  of 
the  eggs  that  form  membranes  segment.     In  the  pres- 


SPECIFICITY  IN  FERTILIZATION  195 

ence  of  the  jelly  many  eggs  form  membranes  bul  fail 
to  segment  owing  to  failure  of  the  spermatozoon  to 
penetrate.  Loeb  suggests  that  the  latter  phenomenon 
may  therefore  be  due  to  agglutination  of  the  starfish 
sperm  to  the  jelly. 

We  gain  here  a  hint  that  will  be  further  developed 
in  the  section  on  the  mechanism  of  fertilization,  viz., 
that  the  specific  factor  in  fertilization  may  concern  an 
agglutination  reaction  between  egg  and  sperm,  as  the 
writer  earlier  maintained. 

In  1906  Godlewski  attempted  to  fertilize  sea  urchin 
eggs  with  sperm  of  starfish,  holothurians,  and  brittle 
stars  by  Loeb's  method  without  success.  However,  he 
succeeded  in  fertilizing  eggs  of  the  same  genera  of  sea 
urchins  with  the  sperm  of  Antedon  rosacea  (crinoid)  by 
the  same  method.  The  fertilizations  succeeded  best  in 
the  alkaline  sea-water  with  a  high  concentration  of 
sperm;  but  some  eggs  were  fertilized  when  first  exposed 
and  then  washed  in  normal  sea-water,  a  fact  that  shows 
the  main  effect  of  the  alkali  to  be  on  the  egg.  A  few 
eggs  might  develop  to  normal  plutei,  thus  exhibiting  a 
purely  maternal  inheritance,  in  spite  of  the  fact  that 
the  sperm  nucleus  fused  with  the  egg  nucleus  and  no 
elimination  of  chromatin  could  be  demonstrated  in 
later  stages. 

Tennent  (19 10)  also  succeeded  in  fertilizing  sea 
urchin  eggs  with  sperm  of  different  echinoderm  classes. 
Thus  the  eggs  of  Hipponoe  were  fertilized  with  the 
sperm  of  Ophiocoma  (brittle  star)  and  of  Pentaceros 
(starfish),  and  the  eggs  of  Toxopneusles  with  the  sperm 
of  Holothuria.  The  method  employed  was  to  allow  the 
eggs  to  stand  two   to   three  hours  before  adding   the 


196  PROBLEMS  OF  FERTILIZATION 

sperm.  A  slight  cytolysis  of  the  egg  is  presumably 
thus  induced.  The  development  was  highly  abnormal 
in  all  cases. 

4.  Sea  urchin  eggs  have  also  been  crossed  with 
sperm  of  different  phyla.  Kupelwieser  (1909  and  191 2) 
has  made  a  special  study  of  this  problem.  He  investi- 
gated the  effect  of  the  sperm  of  fourteen  genera  of 
mollusks  and  annelids  on  sea  urchin  eggs  and  obtained 
positive  but  usually  scanty  results  in  five  cases,  the 
others  being  negative.  A  high  concentration  of  sperm 
and  long  exposure  of  the  eggs  was  necessary.  In  all 
these  cases  membrane  formation  of  the  egg  might  also 
be  induced  by  dead  sperm  or  blood  of  the  species. 
Strongylocentrotus  ?  X  Mytilus  $  gave  the  best  results. 
The  success  of  the  fertilization  seemed  to  depend  on 
extract  present  with  the  sperm,  which  so  affected  the 
surface  of  the  egg  that  one  or  more  spermatozoa 
could  enter.  But  if  membrane  formation  occurred  too 
rapidly,  as  a  result  of  the  sperm  extract  action,  the 
sperm  did  not  enter,  and  the  eggs  died.  Once  within 
the  egg,  if  the  condition  was  monospermic,  events 
moved  normally  to  a  certain  stage;  an  aster  formed  in 
association  with  the  sperm  nucleus;  it  then  formed  an 
amphiaster  while  the  germ  nuclei  united.  The  male 
nucleus  did  not,  however,  form  normal  chromosomes 
and  was  eliminated;  but  the  female  nucleus  formed  its 
chromosomes,  which  divided  in  the  usual  way,  and  all 
nuclei  were  henceforward  haploid  and  purely  maternal. 
In  a  very  small  percentage  of  cases  development  might 
proceed  to  the  pluteus  stage,  which  was  usually  defec- 
tive. It  was  purely  maternal  as  far  as  it  went.  In  the 
very  usual  event  of  dispermy  or  polyspermy  the  phe- 


SPECIFICITY  IN  FERTILIZATION  197 

nomena  were  essentially  similar  to  (lisi)crni>-  or  poly- 
spermy within  the  species:  aster-formation  from  each 
sperm  nucleus,  a  tetraster  or  polyaster  first  cleavage, 
abnormal  development,  and  early  death.  The  lack  of 
specificity  in  the  events  between  penetration  and  clea\-- 
age  is  thus  clearly  shown.  Kupelwieser,  however, 
concludes  that  all  kinds  of  spermatozoa  possess  the 
same  chemical  stuff  for  activation  of  eggs,  an  erroneous 
conclusion  which  we  shall  now  consider. 

Discussion:  These  rapidly  reviewed  data  on  echi- 
noderm  hybridization  demonstrate  in  an  entirely  con- 
vincing manner  the  existence  of  non-specific  factors  in 
fertilization;  and  they  also  demonstrate  with  equal 
clearness  the  existence  of  specific  factors.  The  latter 
are  found  first  in  the  cortical  reactions,  which  never 
occur  with  equal  facihty  in  crosses  outside  the  genus, 
and  second  in  the  latest  stages  of  fertilization  after 
union  of  the  germ  nuclei.  Apparently  any  spermato- 
zoon that  has  once  crossed  the  barrier  of  the  egg  cor- 
tex calls  forth  the  same  set  of  events  within  the  egg. 
The  sperm  aster  is  evidently  a  non-specific  reaction, 
and  when  this  system  is  once  set  in  operation  it  can 
continue  in  only  one  way  so  long  as  it  is  not  impeded 
by  incompatibihties  of  another  kind. 

As  to  the  specific  factors  there  is  unqueslionablv 
resistance  at  the  periphery  of  the  egg,  which  is  most 
promptly  and  readily  overcome  by  the  species  sperm. 
In  the  interspecific  crosses  of  sea  urchins  there  is  not  a 
strong  cortical  resistance,  but  the  quantitative  studies 
necessary  for  an  evaluation  of  the  specific  factor  have 
not  been  made  in  this  case.  When  we  come  to  inter- 
generic  and  wider  crosses  within  the  order  we  find  that 


1 98  PROBLEMS  OF  FERTILIZATION 

the  cortical  resistance  to  the  hybrid  fertiHzation  must 
be  broken  down  by  staling  of  the  eggs  or  dilution 
of  the  sea-water,  or  by  modification  of  the  chemical 
environment,  or  by  high  concentration  of  sperm.  Inter- 
class  crosses  require  in  all  cases  apparently  the  action 
of  some  foreign  agent  on  the  egg;  and  the  same  is 
true  of  the  interphylum  crosses  considered,  though  in 
this  case  the  concentrated  sperm  may  itself  exert  a 
cytolytic  action  on  the  egg,  which  favors  penetration. 

The  specific  factor  that  appears  at  the  end  of  the 
fertilization  process  evidences  itself  usually  in  ehmina- 
tion  of  chromatin;  but  it  is  readily  conceivable  that 
such  a  result  may  not  be  evident;  the  fertilization 
might  to  all  appearance  be  perfectly  normal,  and  yet  sub- 
sequent events  might  demonstrate  the  incompatibility  of 
the  union. 

The  nature  of  these  specificities  need  not  concern 
us  here,  as  they  can  be  considered  more  profitably 
after  other  data  have  been  considered. 

b)  Teleosts:  A  great  many  experiments  have  been 
carried  out  in  the  cross-fertilization  of  various  species 
of  teleosts,  between  species,  genera,  families,  and  orders. 
Thus  Newman  (191 5)  records  seventy-eight  heterogenic 
crosses  between  members  of  different  families  or  orders 
of  teleosts  belonging  to  fourteen  species,  and  Moenk- 
haus  (19 10)  records  eighteen. 

Every  cross-fertilization  attempted  was  more  or  less 
successful  in  the  sense  that  some  or  even  a  large  per- 
centage of  the  eggs  segmented;  no  artificial  treatment 
of  the  eggs  or  the  spermatozoa  appeared  to  be  neces- 
sary in  order  to  secure  these  results,  unlike  echinoderm 
crosses.     Even  in  the  most  distant  heterogenic  crosses 


SPECIFICITY  IN  FERTlLIZAriON  199 

development  might  proceed  to  a  late  stage.  Sooner  or 
later,  however,  the  heterogenic  hybrids  proved  to  be 
non-viable. 

There  is,  however,  according  to  Newnian,  delinite 
evidence  of  specificity  in  teleosts  in  the  sense  that 
species  fertilization  succeeds  much  more  readilx'  than 
any  hybrid  fertilization.  The  percentage  of  hybrid  fer- 
tilized eggs  is  always  less  under  given  conditions  and  is 
frequently  extremely  small.  The  more  ready  union  of 
the  species  sperm  must  depend  upon  some  chemical  rela- 
tion between  egg  and  sperm,  more  highly  de\'elope(l 
between  gametes  of  the  same  than  between  gametes  of 
different  species.  This  obviously  operates  at  the  surface, 
because  the  subsequent  events  of  fertihzation  after  pene- 
tration appear  to  proceed  with  equal  facility  whether 
the  sperm  belongs  to  the  same  or  to  a  different  species. 
Moenkhaus  (1910),  on  the  other  hand,  believes  that,  in 
the  case  of  the  teleosts  studied  by  him,  there  is  no  evi- 
dence of  any  specific  adaptation  of  the  egg  for  its  own 
spermatozoon.  No  adequate  test  of  such  a  conclusion 
appears  to  have  been  made.  The  dr)-  method  of  insemi- 
nation usually  employed  for  teleosts  exposes  the  egg  to 
the  highest  possible  sperm  concentration  and  thus  ren- 
ders a  quantitative  examination  of  the  problem  of 
specificity  impossible. 

Moenkhaus  (1904),  Giinther  and  Paula  Hcrtwig 
(1914),  and  Morris  (1914)  found  normal  penetration  of 
the  spermatozoon,  normal  behavior  of  the  germ  nuclei, 
and  no  evidence  of  chromatin  elimination  in  the  hybrid 
fertilization  of  the  teleosts  that  they  have  studied  cyto- 
logically.  However,  Pinney  (1918)  reports  chromatin 
elimination  in  certain  teleost  crosses  in  the  first  and 


200  PROBLEMS  OF  FERTILIZATION 

second  cleavages,  though  it  did  not  occur  in  the  recip- 
rocal crosses  of  the  same  cases. 

Newman  could  find  no  relation  whatever  between 
success  in  development  and  taxonomic  relationship, 
though  Moenkhaus  is  of  the  contrary  opinion.  Some 
of  the  crosses  between  species  of  the  same  genus  exhib- 
ited much  less  success  in  development  than  other 
crosses  between  members  of  different  orders.  The  eggs 
of  some  species  (e.g.,  Fundiilus  majalis)  never  hybrid- 
ize well,  while  those  of  others  do  well  with  the  sperm 
of  all  species  tried  (e.g.,  Tautoglahrus).  Similarly  the 
sperm  of  some  species  is  better  adapted  to  hybridiza- 
tion than  that  of  other  species.  There  is  a  frequent 
marked  difference  in  the  success  of  reciprocal  crosses, 
but  there  are  notable  exceptions.  In  Newman's  opin- 
ion the  factors  concerned  in  success  or  failure  of  hybrid 
development  of  teleosts  are  associated  with  amount, 
composition,  and  density  of  yolk,  hardiness  or  delicacy 
of  the  species  concerned,  and  certain  mechanical  advan- 
tages or  disadvantages.  Chemical  specificity  thus  ap- 
parently does  not  play  a  leading  role,  though  I  believe 
it  would  be  a  mistake  to  assume  that  it  is  absent. 

c)  Amphibia:  Hybridization  in  Amphibia  has  been 
studied  by  Pfliiger  (1882  and  1883),  Born  (1883  and 
1886),  and  Bataillon  (1906,  1909,  1910)  among  others 
in  a  large  number  of  crosses,  especially  in  the  order 
Anura.  The  hybrid  eggs  never  develop  to  meta- 
morphosis, except  in  the  cases  of  Rana  fusca  6X 
Rana  arvalis  ?  and  Bufo  vulgaris  $  X  Bufo  cinereus  $ 
(Born).  The  other  combinations  die  at  various  stages, 
usually  early.  With  respect  to  fertiHzation  there  is 
immensely  greater  success  than  with  respect  to  viabil- 


SPECIFICITY  IN  FERTILIZATION  201 

ity,  as  is  usual  in  hybrid  c()ml)inations.  The  success 
of  fertiUzation  seems  to  be  entirely  unrehited  to  system- 
atic relationship.  This  may  be  illustrated  by  the  usual 
difference  in  the  success  of  reciprocal  fertilization; 
even  in  species  of  the  same  genus  a  cross-fertiHzation 
may  succeed  one  way  and  fail  entirely  in  the  reciprocal. 
Thus  Pfluger  reports  that  the  eggs  of  Rana  esc  id  cut  a 
fertilize  readily  with  the  sperm  of  R.  fuse  a,  the  eggs 
dying  in  the  blastula  stage;  but  eggs  of  R.  fuse  a 
can  never  be  fertiHzed  with  the  sperm  of  R.  eseuleuta. 
On  the  other  hand  Rana  eseuleuta  and  Rana  arvalis 
fertilize  reciprocally.  The  eggs  of  Rana  fuse  a  coukl 
not  be  fertilized  with  the  sperm  of  any  other 
anuran  (Pfluger),  and  the  same  is  true  of  the  eggs  of 
Pelodytes  (Bataillon);  but  the  eggs  of  the  latter  can 
be  caused  to  develop  by  the  sperm  of  Triton  alpestris 
belonging  to  the  order  Urodela. 

Born  points  out  that  a  higher  concentration  of 
sperm  is  usually  required  for  cross-fertilizations  than 
for  straight  species  fertilizations.  He  distinguishes 
three  kinds  of  behavior  of  the  gametes  in  the  cross- 
fertilizations:  (i)  No  reaction;  examples:  Rana  arvalis 
$  X  Rana  fusea  ?;  Bombinator  igneus  and  Rana  eseu- 
leuta reciprocal;  Pelodytes  6  X  Rana  arvalis  9.  (2)  In 
a  second  group  fertilization  is  apparently  monospermic 
and  normal;  examples:  Rajia  eseuleuta  X  Rana  arvalis 
reciprocal,  Rana  fusea  ^  X  Bufo  eiuereus  ?.  (3)  In  a 
third  group  of  cases  polyspermy  is  the  rule,  followed 
by  early  death  of  the  eggs;  examples:  Bufo  eiuereus 
$  X  Bufo  vulgaris  $,  Pelodytes  .T  X  Rana  eseuleuta  9. 

Unfortunately  this  material  has  not  been  studied 
cytologically   in   any   systematic    way.     Horn    believes 


202  PROBLEMS  OF  FERTILIZATION 

that  in  his  first  group  the  spermatozoa  fail  to  penetrate 
the  egg  membranes,  and  this  is  probably  so,  but  no 
experiments  have  been  undertaken  to  attempt  to  bring 
about  fertilization  by  artificial  means  in  these  cases. 
Bataillon  found  in  the  case  of  the  eggs  of  Pelodytes 
or  of  Bujo  activated  by  the  sperm  of  Triton  that  the 
latter  had  not  entered  at  all,  but  had  at  most  pierced 
the  cortex  of  the  egg;  he  was  therefore  led  to  inquire 
if  a  similar  piercing  by  a  fine  needle  might  not  bring 
about  the  same  results,  and  in  this  experiment  he  suc- 
ceeded in  producing  complete  parthenogenesis,  as  is 
generally  known. 

It  is  obvious  that  the  explanation  of  the  curious 
results  in  hybridization  of  Amphibia  cannot  be  given 
in  terms  of  chemical  specificity  alone.  Pflliger  con- 
cluded that  in  general  those  spermatozoa  are  most 
successful  in  cross-fertilization  that  have  the  thinnest 
heads  and  sharpest  perforatorium;  and  that  eggs  are 
most  accessible  to  hybridization  when  the  spermatozoa 
of  the  same  species  have  thicker  heads.  He  had  thus 
a  conception  that  was  based  purely  on  the  old  idea 
of  the  mechanical  penetration  of  the  spermatozoon 
into  the  egg.  The  results  on  the  Amphibia  do  not 
exclude  a  certain  amount  of  chemical  specificity.  Fuller 
knowledge  of  the  mechanism  in  these  forms  is  necessary 
for  an  explanation  of  the  results. 

Many  of  the  hybrid  fertilized  eggs  die  in  the  blas- 
tula  stage,  but  some  combinations  at  later  stages. 
This  has  usually  been  attributed  to  an  incompatibility, 
chemical  or  otherwise,  between  the  hybrid  chromatins. 
This  idea  has  been  supported  in  an  interesting  way  by 
O.  Hertwig  (1913)  and  G.  Hertwig  (1913).     The  basis 


SPECIFICITY  IN  FERTILIZATION  203 

for  the  experiment  was  the  demonstration  that  exjK)- 
sure  of  spermatozoa  to  radium  emanations  injured  the 
sperm,  and  that  it  might  be  so  graded  as  to  leave  sper- 
matozoa with  ability  to  activate  the  eggs  but  to  transfer 
no  hereditary  effect.     Paula  Hertwig  (1913)  showed  that 
this  was  due  to  failure  of  the  injured  sperm  nucleus  to 
take  part  in  the  cleavage;    hence  such  fertilization  is  a 
kind  of  parthenogenesis,  as  O.  Hertwig  had  previously 
assumed.     O.  Hertwig  showed  that  in  the  cross  Triton 
?    X  Salamandra  S  the  eggs  die  in  the  blastula  stage, 
but  if  the  sperm  be  first  strongly  radiated  the  eggs  will 
produce  larvae  which  possess  the  haploid  number  of 
chromosomes.     This  shows  that  egg  chromatin  alone 
was   concerned   in   the   development   and   permits   the 
inference  that  the  early  death  in  hybrid  fertilization 
with  normal  sperm  is  due  to  the  multiplication  of  the 
sperm  chromatin.     G.  Hertwig  made  a  similar  deter- 
mination for  the  cross  Bufo  vulgaris  ?  X  Rana  fusca  ^ 
Bataillon  (1909)  showed  that  in  fertilization  of  Pelo- 
dytes  ?  by  Triton  the  sperm  nucleus   takes  no  part  in 
cleavage;  nevertheless  the  eggs  die  in  the  blastula  stage, 
owing,  evidently,  to  some  other  cause  than  multiplica- 
tion of  the  sperm  chromatin. 

2.  Self-fertilization. — As  contrasted  with  our  sur\'e\' 
of  hybrid  fertilization  we  should  next  consider  the  data 
concerning  the  self-fertilization  of  hermaphrodite  organ- 
isms, i.e.,  the  fertilization  of  the  eggs  by  the  spermato- 
zoa of  the  same  individual.  If  dissimilaritv  of  gametes 
is  the  cause  that  renders  hybrid  fertiHzation  difficult, 
it  might  be  expected  that  the  closest  possible  relation- 
ship of  gametes,  which  is  found  in  hermaphrodite  indi- 
viduals, would  involve  the  greatest  compatibility  of  the 


204  PROBLEMS  OF  FERTILIZATION 

gametes.  But,  as  is  well  known,  this  is  by  no  means 
always  the  case,  for  there  are  both  hermaphrodite 
animals  and  plants  in  which  self-fertilization  is  difficult 
or  impossible.  Two  problems  have  usually  been  con- 
sidered together  in  this  connection,  viz.,  the  problem 
of  compatibiHty  in  fertilization  and  the  problem  of 
viability  and  vigor  of  the  offspring  of  such  fertiliza- 
tions. The  latter  problem,  although  related,  will  not 
concern  us  here. 

The  problem  of  self-fertilization  has  not  been  very 
widely  investigated  in  the  case  of  hermaphrodite  an- 
imals. In  rhabdocoel  Turbellaria  reproduction  by  self- 
fertilization  is  common;  it  is  also  stated  to  occur 
occasionally  in  certain  trematodes  and  cestodes  in  spite 
of  an  elaborate  apparatus  for  cross-fertihzation.  Ohgo- 
chaetes  and  pulmonates  appear  to  reproduce  exclusively 
by  cross-fertilization;  but  Braun  (1888)  and  Colton 
(191 2)  have  shown  that  individuals  of  the  pond  snail 
Limnaea  reared  in  isolation  from  the  egg  may  produce 
fertile  eggs.  As  parthenogenesis  is  unknown  in  mol- 
lusks  it  is  almost  certain  that  these  eggs  were  self- 
fertihzed.  A.  H.  Cook  reports  a  similar  case  for  Arion 
(Cambridge  Natural  History).  In  the  parasite  cirripeds 
(Rhizocephala)  reproduction  is  invariably  by  self- 
fertilization  (J.  W.  Smith,  1906),  and  the  same  is  true  of 
certain  free-living  nematodes  (Maupas,  1900;  Potts, 
1910).  In  both  of  the  latter  groups  special  arrange- 
ments  exist  for  insuring  self-fertihzation.  Among  the 
ascidians  Cynthia  and  Molgula  appear  to  be  self-fertile, 
at  least  to  a  considerable  extent  (Morgan,  1904),  but 
Ciona  in  the  same  group  is  self-infertile,  at  least  to  a 
considerable  extent,  which  appears  to  vary  somewhat 


SPECIFICITY  IN  FERTILIZATION  205 

for  different  individuals  and  localities  (Caslle,  190,^; 
Morgan,  1904,  1905,  1910;   Fuchs,  19 14,  191 5). 

In  plants  the  problem  of  self-fertilization  was 
brought  to  the  forefront  of  investigation  by  Darwin's 
classical  ''Studies  on  Cross-  and  Self-Fertilization  in 
the  Vegetable  Kingdom,"  and  the  problem  of  selfing 
has  been  very  carefully  and  extensively  studied  in 
recent  years  by  Jost  (1907),  Correns  (191 2),  East 
(1915(7,  1915^),  East  and  Parke  (1917),  Stout  (1916 
and  191 7),  and  others.  The  problem  in  plants  is  simi- 
lar in  many  respects  to  that  in  animals,  but  it  should 
be  remembered  that  in  plants  the  incompatibility  that 
occurs  in  many  cases  concerns  the  growth  of  the  pollen 
tube,  which  is  more  or  less  abortive  on  the  stigma  and 
in  the  style  of  the  same  flower  in  such  cases,  and  not 
the  reaction  of  the  actual  gametes,  which  appear  usually 
not  to  meet.  Of  course  in  many  plants  the  pollen  of  the 
same  flower  is  perfectly  compatible,  and  in  the  case  of 
cleistogamous  flowers  that  never  open  but  nevertheless 
produce  perfect  seed  there  is  no  chance  for  cross- 
fertilization.  The  phenomenon  of  physiological  incom- 
patibility of  own  pollen  is  more  or  less  sporadic  in  its 
occurrence,  and  in  fact  the  plants  in  which  this  occurs 
form  a  relatively  small  class. 

The  ascidian  Ciona  is  the  only  known  and  carefully 
studied  example  of  physiological  self-incompatibility  of 
gametes  in  the  animal  kingdom.  1'he  various  authors 
who  have  studied  this  case  (Castle,  ]\Iorgan,  Fuchs) 
all  found  certain  individuals  in  which  the  eggs  arc  not 
susceptible  of  fertihzation  with  the  sperm  (^f  the  same 
individual,  although  they  may  be  fertilized  with  sperm 
of  other  individuals;    and  the  si)erm  thus  impotent  on 


2o6  PROBLEMS  OF  FERTILIZATION 

eggs  of  the  same  individual  may  fertilize  perfectly  the 
eggs  of  other  individuals.  The  failure  to  self-fertihze 
in  these  cases  is  not  due  to  immobility  of  the  spermat- 
ozoa in  the  presence  of  own  eggs,  or  inability  to  reach 
the  membrane  of  the  egg,  but  it  is  due  to  absence  of 
the  reaction  that  leads  to  penetration  of  the  egg  by  the 
spermatozoon.  Such  incompatibility  is  by  no  means 
universal  in  Ciona,  for  all  authors  have  found  certain 
individuals  in  which  self-fertilization  may  occur  to  a 
certain  extent. 

The  determination  of  the  occurrence  of  self- 
fertilization  obviously  requires  much  care  to  avoid  con- 
tamination with  the  sperm  of  other  individuals.  The 
method  which  was  originally  employed  by  Castle  in 
his  determinations  consisted  in  comparing  the  percent- 
ages of  fertilized  eggs  from  isolated  individuals  with 
the  percentages  from  pairs  of  individuals  placed  to- 
gether. Observations  were  made  on  the  same  individ- 
uals for  five  successive  days,  and  the  fertilized  eggs  of 
each  day  were  separately  estimated.  The  result  was 
that  of  fifty  estimates  from  ten  isolated  individuals 
thirty-seven  contained  no  eggs  fertilized,  nine  from  4 
per  cent  to  25  per  cent  fertilized,  two  contained  90 
per  cent  of  fertilized  eggs,  and  in  two  cases  no  eggs 
were  deposited.  The  paired  individuals  yielded  twenty- 
five  estimates,  of  which  twenty-three  showed  100  per 
cent  fertilized,  one  yielded  20  per  cent,  and  one  none 
fertilized. 

To  this  method  of  determining  the  extent  of  self- 
fertihzation  the  objection  has  been  made  that  spermat- 
ozoa of  foreign  origin  may  remain  in  the  atrial  cavity 
or  tangled  in  the  branchial  basket  and  give  the  effect 


SPECIFICITY  IN  FERTILIZATION 


207 


of  self-fertilization  when  none  exists.  But  Fuchs  has 
shown  that  shed  spermatozoa  will  not  survive  over 
twenty-four  hours  in  sea- water,  so  that  the  tests  arc 
probably  valid  for  determinations  after  twenty-four 
hours  of  isolation,  on  the  assumption  that  in  the  case 
of  pairs  both  individuals  shed  their  gametes.  Castle 
himself  suspects  contamination  in  the  two  cases  of  appar- 
ent self-fertilization  yielding  90  per  cent  of  fertilized  eggs. 

To  avoid  this  objection  artificial  insemination  has 
been  practiced  by  the  three  authors  named.  In  these 
experiments  Morgan  finds  an  almost  vanishing  amount 
of  self-fertilization;  while  Fuchs  working  in  Naples 
found  that,  while  in  many  cases  no  eggs  segmented 
after  self-fertilization,  nevertheless  Ciona  intestijialis  as 
a  species  is  far  from  being  completely  self-sterile,  though 
''a  greater  concentration  of  sperm  is  usually  necessary 
to  bring  about  any  self-fertilization  than  would  cross- 
fertihze  100  per  cent  of  foreign  eggs." 

May  self-fertilization  be  forced  on  these  eggs  ? 
Fuchs  found  that  by  increasing  the  quantit}'  of  sperm 
a  higher  percentage  of  self-fertilization  could  be  secured, 
as  shown  in  the  following  table: 


Eggs  Selfed 

Eggs  Crossed 

5  Drops 
Sperm 

4  c.c.  Sperm 

5  Drops 
Sperm 

4  C.C.  Sperm 

Specimen  A, . . 

0 

0 

12 

0 

5'S 

22 

100 

56 

100 
100 

TOO 
100 

TOO 

Specimen  B 

100 

Specimen  C 

100 

Specimen  D 

100 

Equal  amounts  of  eggs  of  four  specimens  were  added 
to  four  equal  quantities  of  sea-water,  and  the  first  two 


2o8  PROBLEMS  OF  FERTILIZATION 

dishes  of  each  were  selfed  and  the  second  two  of  each 
crossed  with  quantities  of  sperm  shown.  The  results 
are  expressed  as  percentages  of  eggs  segmenting.  It 
will  be  observed  that  the  lesser  quantity  of  sperm  suf- 
ficed in  cross-fertilization  to  cause  the  segmentation  of 
every  egg,  but  was  without  any  effect  in  the  case  of  three 
of  the  four  self-fertilizations,  and  fertilized  only  12  per 
cent  in  the  case  of  the  other ;  the  higher  concentration  of 
sperm  caused  some  self-fertilization  in  every  case  recorded 
in  the  table,  though  it  seems  probable  from  other  state- 
ments that  this  is  not  always  the  case.  Fuchs  also 
determined,,  as  I  interpret  his  results,  that  staling  of 
the  eggs  in  sea-water  increases  their  susceptibility  to 
self-fertilization  up  to  a  certain  point;  this  was  in 
marked  contrast  to  cross-fertilization.  Morgan  has 
attempted  to  bring  about  self-fertilization  by  the  action 
of  ether,  alcohol,  chlorotone,  and  other  substances,  with 
results  that  must  in  general  be  regarded  as  negative, 
for  they  were  often  contradictory. 

There  appears  to  be  a  certain  general  resemblance 
between  resistance  to  hybrid  and  to  self-fertilization, 
because  the  same  conditions  may  overcome  either;  it 
must,  however,  be  admitted  that  the  data  on  the  self- 
fertilization  are  rather  scanty  and  inconsistent  in  this 
particular.  The  evidence  is,  nevertheless,  adequate  to 
prove  that  the  resistance  is  cortical  in  the  one  case  as 
in  the  other,  and  that  when  this  cortical  resistance  is 
overcome  the  internal  events  of  fertilization  proceed 
normally. 

Are  the  eggs  of  Ciona  equally  fertile  to  the  sperm 
of  all  other  individuals,  or  is  there  a  certain  degree  of 
individual  as  well   as  of  self-incompatibiHty  ?     In   an 


SPECIFICITY  IX  FERTILIZATION 


20Q 


extensive  set  of  trials  involving  over  six  hundred  fertil- 
izations Morgan  found  very  considerable  variations  in  the 
percentages  of  fertilization  in  different  combinations. 
The  experiments  were  usually  conducted  with  groups  of 
four  or  six  individuals  and  involved  all  reciprocals  and 
the  self-fertilizations  in  each  case.  While  all  the  results 
cannot  be  regarded  as  equally  free  from  error,  they 
nevertheless  justify  the  conclusion,  when  taken  together, 
that  there  are  verv  considerable  variations  with  refer- 
ence  to  fertilizing  power  in  different  combinations,  even 
reciprocal  ones,  in  spite  of  Fuchs's  criticism  of  this 
result. 

It  may  be  pointed  out  that  the  problem  of  indi- 
vidual incompatibility  is  not  necessarily  associated  with 
hermaphroditism  and  the  problem  of  self-sterility;  it 
is  probable  that  there  is  a  considerable  amount  of  indi- 
vidual variation  with  respect  to  gamete  compatibility 
in  species  with  separate  sexes,  though  little  is  known  on 
this  point. 

Even  when  self-fertilization  succeeds,  the  viability 
of  the  resulting  larvae  is  relatively  slight,  although,  as 
will  be  seen  from  the  following  table  (after  F'uchs),  the 


9  w 

Time 

Time  of 

2   N 

OF  4 

Hatch- 

Alive 

Sri 

Cells 

ing 

Settled 

Perci 
Pert 

Down 

Min. 

Hr. 

Min. 

8  Days 

20  Days 

Self-fertilized  . .  |g^ 

7 
<2 

86 
83 

19 
19 

37   Very  few 
30   More 

None 
None 

None 
None 

^Ab 

ICO 

83 

IQ 

15  'Most 

Equal 

Fewest  of 

Cross-fertilized  • 

Ba 

ICO 

8,S 

19 

27 

Many 

number 

.\b,  med- 

Ca 

IOC 

86 

19 

26 

Most 

of  .\b  and 

umi   num- 

Ba,  more 
of  Ca 

ber  of  Ba, 
most  of  Ca 

2IO  PROBLEMS  OF  FERTILIZATION 

rates  of  development  are  about  the  same  for  self-  and 
cross-fertilized  eggs. 

In  this  table  each  letter  stands  for  an  individual, 
the  capital  for  the  eggs  and  the  small  letter  for  the 
sperm;  thus  Aa,  Bb  are  self-fertilizations,  Ab,  Ba,  and 
Ca  cross-fertilizations.  An  excess  of  sperm  was  used  in 
all  the  fertilizations;  the  difference  in  percentage  of 
eggs  fertilized  when  selfed  and  crossed  should  be  noted. 
The  eggs  that  are  fertilized  segment  and  hatch  in  the 
same  time,  whether  selfed  or  crossed;  but  the  selfed 
eggs  do  not  survive,  while  the  crossed  eggs  do.  This 
was  the  invariable  result  in  a  considerable  number 
of  experiments,  except  that  in  one  selfed  lot  some  of 
the  larvae  settled  down,  and  four  survived  over  a 
month. 

It  is  clear  from  the  discussion  that  the  incompati- 
bihty  of  the  self-sperm  manifests  itself  in  the  cortical 
reaction;  for  some  reason  the  spermatozoon  fails  to 
fuse  with  the  egg,  and  as  a  consequence  the  activation 
of  the  egg  fails.  This  result  is  incidentally  another 
strong  proof  that  the  process  of  union  of  the  gametes  is 
not  a  mechanical  boring-in  action  of  the  spermatozoon. 
The  experiments  may  also  be  regarded  as  demonstrat- 
ing that  if  the  cortical  barrier  is  once  passed  the  other 
processes  of  fertihzation  proceed  normally. 

Morgan  (1910)  in  his  analysis  of  the  subject  con- 
cludes that  the  failure  to  self-fertilize  is  due  to  the 
absence  of  a  reaction  between  the  egg  and  the  sperm; 
and  elsewhere  (19 13)  he  attributes  this  to  the  similarity 
of  the  hereditary  factors  carried  by  the  egg  and  the 
sperm.  The  context  of  his  analysis  implies  that  he 
is   thinking  of  a   chemical   reaction   of   the   character 


SPECIFICITY  IN  FERTILIZATION  211 

of  an  immunity  reaction  in  a  very  general  sense.  'I'his 
implies  that  a  certain  chemical  differentiation  of  the 
gametes  is  necessary  for  the  fertilization  reaction,  and 
that  such  differentiation  may  be  lacking.  It  seems  to 
the  writer  that  this  step  in  interpretation  is  along  the 
right  line,  but  it  is  clear  that  it  needs  to  be  carried 
farther  by  more  investigation.  Nothing  would  probably 
contribute  more  to  a  comprehension  of  the  biochemical 
factors  on  which  the  fertilization  reaction  depends  than 
the  solution  of  the  problem  of  self-sterility. 

The  solution  might  be  carried  a  step  farther  if  we 
were  to  assume  that  the  egg  produces  an  agglutinating 
substance  necessary  to  bind  the  sperm  for  the  cortical 
reaction,  but  that  the  agglutinating  substance  does  not 
operate  on  own  sperm  in  the  case  of  Ciona.  We  have 
seen  in  chapter  iv  that  ova  actually  do  produce  a 
sperm-agglutinating  substance,  and  the  relation  of  this 
to  fertilization  is  discussed  in  the  next  chapter;  the 
foregoing  assumption  is  therefore  not  a  mere  fancy. 

Before  discussing  the  matter  further  let  us  very 
briefly  review  the  analogous  phenomena  in  flowering 
plants,  in  which,  as  we  have  already  stated,  a  relatively 
small  number  exhibit  the  phenomenon  of  physiological 
self-incompatibiHty  in  pollination.  Stout  (1916)  and 
East  (191 7)  have  given  excellent  historical  reviews  of 
the  literature  of  this  subject.  It  should  be  borne  in 
mind  in  comparing  plants  with  animals  that  the 
incompatibility  in  the  former  consists  in  inhibition  of 
growth  of  the  pollen  tube  and  not  incomixitibility  of 
the  actual  gametes.  There  is  no  evidence  that  self- 
steriHty  in  plants  is  ever  due  to  incomi)atibility  of  the 
actual  gametes. 


212  PROBLEMS  OF  FERTILIZATION 

Darwin  has  reported  more  than  thirty  cases  of  self- 
incompatibihty,  sometimes  absolute,  sometimes  only 
relative;  since  then  numerous  additional  cases  have 
been  cited.  The  genera  most  carefully  studied  have 
been  Corydalis,  Secale,  Lilium,  Cardamine,  Antirrhinum, 
Reseda,  Nicotiana,  and  Cichorium.  Self-sterility  is  prob- 
ably never  absolute  in  any  species;  it  may  appear  in 
normally  self-fertile  species  sporadically  (Stout),  or  a 
self-sterile  individual  may  become  self-fertile  under  ad- 
verse conditions  (East  and  Park,  191 7).  Pollination  of 
different  flowers  of  the  same  plant  (geitonogamy)  may 
be  slightly  more  successful  than  strict  self-fertilization 
(autogamy),  but  not  always;  in  Lilium  bulbiferum  all 
the  plants  of  the  same  clone  have  been  found  to 
be  cross-sterile,  but  seed-sisters,  on  the  other  hand, 
cross-fertile.  The  phenomenon  may  thus  concern  self- 
sterility  of  parts  of  the  same  flower,  sterility  between 
different  flowers  of  the  same  plant,  and  sterility  between 
asexually  produced  offspring  of  the  same  plant.  Recent 
experiments  (Correns,  Stout,  East)  have  also  shown 
that  it  may  be  transmitted  like  Mendelian  characters 
and  thus  affect  in  the  form  of  cross-sterility  entire 
sections  of  a  population. 

The  study  of  this  subject  entered  a  new  phase  with 
Correns'  experiments  on  the  inheritance  of  self -infertility. 
He  took  two  unrelated  plants  B  and  G  of  Cardamine 
pratensis,  which  were  self-sterile,  and  made  the  recip- 
rocal crosses  B?  X  G6  and  G?  X  B6,  constituting  series 
I  and  2  respectively,  and  then  investigated  (i)  the 
relations  of  the  parents  and  offspring  to  two  unrelated 
plants,  (2)  the  relations  of  the  pollen  of  both  parents 
to  thirty  offspring  of  each  series,  and  (3)  the  relations 


SPECIFICITY  IN  FERTILIZATION  213 

of  all  possible  combinations  of  the  offspring  to  one 
another.  The  third  set  of  experiments  could  not  of 
course  be  fully  carried  out. 

The  parents  and  offspring  were  shown  to  be  recip- 
rocally compatible  with  the  two  unrelated  plants.  In 
the  second  set  of  experiments  he  found  that  the  off- 
spring are  divisible  into  two  approximately  equal  classes 
for  each  parent,  viz.,  fertile  with  the  parent  or  sterile, 
including  in  the  second  class  some  that  set  seed  very 
scantily.  The  relation  of  a  given  offspring  to  one 
parent  is  entirely  independent  of  its  relation  to  the 
other.  The  offspring  may  therefore  be  divided  into 
four  classes,  viz.: 

1.  Fertile  with  both  parents  B  and  G  =  bg. 

2.  Fertile  with  parent  B,  sterile  with  G  =  bG. 

3.  Fertile  with  parent  G,  sterile  with  B  =  Bg. 

4.  Sterile  with  both  parents=BG 

The  stated  result  obviously  indicates  heritable  char- 
acters concerned  in  self-incompatibility.  Correns  as- 
sumes that  these  are  to  be  interpreted  as  stuffs  that 
inhibit  the  normal  development  of  self-pollen,  whether 
in  a  positive  sense  or  in  the  sense  of  the  absence  of  a 
stuff  necessary  to  growth  of  the  pollen  tube.  If  this 
is  so,  the  behavior  of  the  offspring  among  themselves 
with  reference  to  self-sterility  should  be  predicable,  and 
Correns  states  from  the  results  of  crosses  of  twelve  of 
the  offspring  with  all  of  the  others  that  the  expect  at  icm 
given  by  the  formulae  is  approximately  realized.  He 
argues  that  the  demonstration  of  the  heritability  of 
incompatibility  disproves  Jost's  theory  that  the  selt- 
incompatibility  is  due  to  individual  stuffs;  that  is,  to 
the  chemical  make-up  of  each  individual  being  sj^ccific; 


214  PROBLEMS  OF  FERTILIZATION 

but  he  agrees  as  to  the  chemical  foundation  of  the 
phenomenon;  each  stuff  depends  on  a  gene  in  the  germ 
plasm,  and  it  is  the  combination  of  stuffs  that  is  formed 
by  each  gametic  union  that  is  specific  or  individual. 
He  thus  bases  the  phenomenon  on  line  combinations  of 
stuffs.  Correns  must  receive  the  credit  of  being  the  first 
to  relate  self-sterility  to  cross-sterility  within  the  species. 
Stout  (191 6,  191 7)  has  carried  out  a  very  elaborate 
and  finely  executed  series  of  experiments  on  Cichorium. 
In  a  combination  similar  to  Correns  he  made  sixty- 
nine  back  crosses  of  offspring  on  a  known^  parent,  of 
which  thirty-five  were  fertile  and  thirty-four  sterile, 
thus  agreeing  with  Correns'  ratios.  He  observes  that 
''the  range  of  variation  in  the  actual  fertility,  how- 
ever, is  so  great  in  both  cases  that  the  grouping  of 
all  offspring  into  two  classes  with  reference  to  cross- 
fertility  with  a  parent  is  of  little  significance."  He 
confirms  in  general  the  relation  of  self-  and  cross- 
sterility  within  the  species  which  Correns  discovered, 
but  believes  that  Correns'  conception  of  line  stuffs  is 
fundamentally  wrong.  As  demonstration  of  this  he 
shows  that  self-fertile  plants  may  arise  in  certain 
crosses  between  self-sterile  individuals;  when  these 
self-fertile  offspring  of  the  Fj  generation  were  selfed  he 
obtained  forty-one  self-sterile  and  thirty-nine  self-fertile 
plants  in  F^;  in  F3  of  selfed  plants,  self-fertile  and  self- 
sterile  plants  arose  again  in  about  equal  numbers.  He 
also  shows  that  neither  self-sterility  nor  self-fertility  is  a 
dominant  or  recessive  character  in  any  consistent  sense, 
"and  there  is  a  very  irregular  and  sporadic  inheritance 
both  of  the  character  as  such  and  the  degree  of  its 
expression,  if  the  two  can  in  any  sense  be  separated." 


SPECIFICITY  IN  FERTILIZATION  215 

East  and  Park  (191 7),  however,  demonstrate  very 
conclusively  in  Nicotiana  that  ''self-sterility  is  a  condi- 
tion determined  by  the  inheritance  received,  but  can 
develop  to  its  full  perfection  only  under  a  favorable 
environment."  Thus  at  the  end  of  a  flowering  period 
and  under  conditions  adverse  to  vegetative  growth,  a 
certain  amount  of  self-fertility  may  obtain  in  plants 
that  are  entirely  self-sterile  at  other  times.  The  extent 
of  this  change  varies  with  the  species;  but  the  offspring 
arising  from  such  self-fertilization  are  self-sterile,  a  fact 
that  demonstrates  the  fluctuating  character  and  envi- 
ronmental origin  of  the  self-fertilization  to  which  the 
oft'spring  owe  their  existence.  Such  self-fertility  does 
not  differ  from  other  cases  of  suppression  of  characters 
by  environment.  When  this  principle  was  borne  in 
mind  the  results  of  the  extensive  experiments  became 
intelligible  and  permitted  a  consistent  genetic  analysis. 

The  view  of  Darwin  that  too  great  uniformity  of 
organization  of  the  gametes  operates  to  prevent  success- 
ful fertilization  recurs  in  one  form  or  another  up  to  the 
present  time.  Darwin  cited  the  infertility  that  some- 
times arises  as  a  result  of  inbreeding  as  an  analogous 
phenomenon.  It  is  obvious  that  Jost's  and  Correns' 
interpretations  also  rest  on  the  ground  of  too  close  rela- 
tionship, assumed  by  them  to  be  chemical,  and  related 
by  Correns  to  heritable  genes.  East  assumes  the  neces- 
sity for  the  secretion  of  stimulating  substances  by  the 
pistils  for  the  growth  of  the  pollen  tube  that  can  be 
''called  forth  only  by  a  gamete  that  differs  from  the 
somatic  cells  between  which  the  pollen  tube  passes'' 
(quoted  from  Stout,  1916).  Morgan  (1913)  holds  that 
"the  failure  to  self- fertilize,  which  is  the  main  problem, 


2i6  PROBLEMS  OF  FERTILIZATION 

would  seem  to  be  due  to  the  similarity  in  the  hereditary 
factors  carried  by  eggs  and  sperm."  Stout  on  the  other 
hand,  arguing  from  hybrid  incompatibilities,  maintains 
that  "the  most  fundamental  principle  of  sexual  fertility 
is  that  a  marked  degree  of  similarity  in  constitution  is 
necessary"  for  the  existence  of  compatibiHty.  He  is 
thus  inclined  to  refer  self-incompatibility  to  constitu- 
tional dissimilarity  between  gametes,  which  must 
therefore  be  strictly  of  epigenetic  origin.  He  cannot, 
however,  deny  the  occurrence  of  a  certain  amount  of 
cross-incompatibility  as  a  result  of  heredity;  so  that 
his  views  seem  to  lack  consistency  in  this  respect. 

It  is  obvious  that  genetic  studies  cannot  solve  the 
problem  of  self-incompatibility  in  a  physiological  sense; 
on  the  other  hand,  if  a  physiological  solution  were 
found  the  genetic  results  would  be  more  readily  inter- 
pretable.  The  elementary  fact  that  the  gametes  which 
produced  any  hermaphrodite  individual  were  ipso  facto 
compatible,  though  they  may  themselves  transmit 
incompatibility,  proves  that  the  latter  property  is 
cytoplasmic,  belonging  to  the  duplex  organization  and 
not  to  the  genes  of  the  mature  gametes.  Confusion 
between  the  possession  and  transmission  of  incompati- 
bility must  be  avoided. 

East  and  Park  (191 7)  have  shown  that  the  differ- 
ence in  behavior  between  compatible  and  incompatible 
pollen  on  the  stigma  and  in  the  style  is  that  the  compat- 
ible pollen  tube  grows  with  constant  acceleration,  exhib- 
iting an  autocatalytic  curve,  whereas  the  incompatible 
pollen  tubes  grow  at  a  constant  rate  and  hence  fail  to 
reach  the  ovule  before  the  style  withers  and  growth 
becomes  impossible.     They  therefore  assume  that  com- 


SPECIFICITY  IN  FERTILIZATION  217 

patible  pollen  facilitates  the  formation  of  substances 
from  the  cells  of  the  style  that  stimulate  growth. 

If  we  attempt  to  seek  the  explanation  along  the  lines 
of  serum  reactions,  an  obvious  possibility  that  various 
authors  have  considered,  it  must  be  realized  at  the  out- 
set that  for  the  present  only  broad  analogies  can  be 
considered;    if  a  reaction  comparable  to  immune  reac- 
tions is  concerned  in  fertilization  it  may  belong  to  a 
new  category  and  not  to  any  of  the  recognized  cate- 
gories, whether    of    lysins,    agglutinins,   or  precipitins. 
The  question  would  be  whether  a  point  of  view  could 
be  formulated  consistent  with  the  specificities  both  of 
fertilization  and   of  immunology.     The  analogy  would 
naturally  be  with  the  antigen    X    antibody  reactions. 
The  principle  of  the  reaction  may  be  illustrated  thus: 
When  an  animal  receives  repeated  injections  of  the  red 
blood  cells  of  another  species  the  serum  acquires  the 
power  of  dissolving  the  red  blood  cells  of  the  other 
species    (haemolysis);    the    reaction    is   specific   in   the 
sense  both  of  the  species  used  and  also  of  the  kind  of 
cell  used.     In  this  case  the  blood  cells  injected  consti- 
tute the  antigen,  and  the  dissolving  substance  formed 
in  the  blood  of  the  other  animal  the  antibody.     In  this 
case  it  is  found  too  that  another  antibody,  an  agglu- 
tinin, is  also  formed  in  the  serum  of  the  other  species. 
This  is  more  thermostable  than  the  lysin  and  may  there- 
fore be  demonstrated  by  heating  the  immune  serum  to 
56°  C,  which  destroys   the   haemolytic  property,   but 
leaves  the  agglutinating  substance  intact. 

It  has  also  been  found  that  isohaemolysins  and 
isoagglutinins  may  be  devel()i)ed  in  certain  cases  by 
injecting  blood  cells  of  another  indi\'idual  of  the  same 


2i8  PROBLEMS  OF  FERTILIZATION 

species.  But  it  has  hitherto  been  impossible  to  develop 
autohaemolysins  or  autoagglutinins  by  injection  of  the 
broken-down  blood  cells  of  the  same  individual. 

This  statement  will  be  sufficient  for  purposes  of  com- 
parison. The  analogy  to  fertilization  would  rest  on 
the  resemblance  of  isoagglutination,  for  instance,  to 
cross-fertilization,  and  on  the  absence  of  autoaggluti- 
nation  to  the  absence  of  self-fertilization.  But  if  the 
egg  produces  a  specific  antibody  to  the  sperm,  as  the 
analogy  suggests,  this  must  be  based  on  some  substance 
in  the  sperm,  which  acts  as  antigen,  of  different  chemical 
composition  from  the  homologous  substance  of  the  egg. 
Why  then  should  not  the  antibody  develop  equally 
well  in  the  body  of  a  hermaphrodite  as  of  a. female, 
seeing  that  the  postulated  differences  of  gametes  must 
hold  equally  well  for  both  cases  ?  It  is  obvious  that  the 
serum  analogy  breaks  down  here,  and  some  new  principle 
must  be  invoked;  for  instance,  that  antigen  and  antibody 
do  not  react  with  one  another  if  developed  in  the  same 
body.     But  this  merely  restates  the  old  difficulty. 

It  is  obvious  that  the  principles  of  immunity  cannot 
apply  to  fertilization  in  any  such  sense.  All  we  can 
hope  to  utilize  from  immunity  reactions  is  the  fact  of 
the  existence  of  chemical  specificities  of  an  equally 
definite  and  specialized  character.  The  mechanism 
must  be  quite  different  in  the  two  cases. 

3.  Discussion. — If  we  gather  together  our  discussion 
of  specificity  in  fertihzation  it  will  be  seen  that  the  stage 
in  which  the  phenomenon  of  specificity  most  commonly 
manifests  itself,  whether  in  the  hybrid  fertilization  of 
echinoderms,  teleosts,  and  Amphibia  or  in  the  phenom- 
ena of  self-infertility  of  Ciona,  is  in  the  cortical  reactions. 


SPECIFICITY  IN  FERTILIZATION  219 

The  various  methods  used  to  induce  hybrid  fcrtihza- 
tion— staHng  of  eggs,  high  concentration  of  sperm,  use 
of  alkahes  or  other  chemicals — have  therefore  this  one 
feature  in  common,  that  they  destroy  the  chemical  or 
physical  integrity  of  the  cortex  of  the  egg.  Thus  is 
rendered  possible  a  form  of  reaction  common  to  all 
gametes,  the  inclusion  of  the  spermatozoon  within  the 
egg.  Specific  factors  then  do  not  apparently  intervene 
until  after  the  meeting  of  the  germ  nuclei. 

If  there  is  indeed  a  specific  factor  in  the  cortical 
reaction  it  can  hardly  be  of  a  purely  physical  character, 
though  physical  factors  must  be  of  significance  in  any 
case;  for  the  specific  factor  must  include  not  only 
the  incompatibilities  in  hybrid  ferlihzation  but  also 
those  in  self-fertihzation  where  the  physical  resemblance 
of  the  gametes  excludes  a  purely  physical  explanation 
of  the  phenomenon.  We  are  therefore  forced  to  the 
conclusion  that  there  is  a  chemical  specificity,  more  or 
less  narrow,  in  the  union  of  the  gametes.  The  only 
phenomenon  in  which  we  can  so  far  detect  a  closer 
approach  to  this  problem  is  that  of  sperm  agglutination 
by  egg  secretions. 

A  warning  is  in  place  here  against  too  much  reliance 
on  the  phenomenon  of  specific  agglutination  in  the 
field  of  immunology  by  way  of  comparison.  It  may 
turn  out  that  there  is  a  much  closer  relationship  be- 
tween these  phenomena  than  we  can  now  detect,  or 
they  may  represent  only  superficially  similar  phenomena 
with  the  factor  of  specificity  in  common,  and  that  onl}- 
to  a  certain  extent,  at  present  unknown. 

We  have  the  very  striking  fact  reciprocal  to  speci- 
ficity in  fertilization,  that  spermatozoa,  of  certain  forms 


2  20  PROBLEMS  OF  FERTILIZATION 

at  least,  are  agglutinated  together  by  egg  secretions  of 
the  same. species.  This  phenomenon  is  definitely  tissue 
specific,  as  I  have  already  pointed  out.  At  the  present 
time  the  extent  of  specificity  between  species  has  been 
inadequately  investigated.  This  subject  has  been  dis- 
cussed in  chapter  iv,  and  it  would  appear  that  there  may 
be  a  sufiicient  degree  of  specificity  there  to  account  for 
the  specificities  in  fertilization,  with  the  exception  of 
self -infertility,  which  has  not  been  investigated  from 
this  point  of  view.  The  relation  of  sperm  agglutina- 
tion to  the  fertilization  reaction  itself  is  considered  in 
chapter  vii. 

While  it  is  by  no  means  certain  that  specificity  in 
fertilization  depends  upon  specific  agglutination  of  the 
spermatozoon  to  the  egg,  yet  I  think  it  must  be 
admitted  that  the  latter  phenomenon  furnishes  an  - 
important  clue  for  the  further  analysis  of  the  problem 
of  specificity  in  animals.  Until  this  is  made,  the 
temptation  to  speculate  further  along  these  fines  should 
better  be  resisted. 


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SPECIFICITY  IN  FERTILIZATION  223 

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1913-  ''Das  VerhaUeii  des  mil  Radium  bestrahlten  sperma- 
chromatins  im  Froschei,"  Arch,  fiir  mikr.  Anat.  u. 
Entwicklungsgesch.,  Band  81,  Abt.  II,  pp.  iu-^2. 

JOST,   L. 

1907.  "Ueber  Selbststerilitiit.  einiger  Bluten,"  Botan. 
Zeilung.,  Heft.  V  und  \T,  p.  112. 

KOHLBRUGGE,   J.    H.    F. 

1910.  ''Der  Einfluss  der  Spermatozoidcn  auf  die  Blastula," 
Arch,  fiir  mikr.  Anat.  u.  Entwicklungsgesch.,  Band  75. 

1910-11.  "Der  Einfluss  der  Spermatozoiden  auf  den  Uterus; 
ein  Beitrag  zur  Telegonie,"  Zeitschr.  fiir  Morph.  u. 
Anthropol.,  Band  12,  pp.  359-68;  Band  13,  pp.  19-30. 

191 2.  ''Die  Verbrcitung  der  Spermatozoiden  im  weiblichen 
Korper  und  im  befruchteten  Ei,"  Arch,  fiir  Ent- 
wickelungsmech.,  Band  35,  pp.  165-88. 

KuPELwiESER,  Hans. 

1909.  ''Entwicklungserregung  bei  Seeigeleiern  durch  :\rol- 
luskensperma,"  Arch,  fiir  Entwickelimgsmech.,  Band 
27,  pp.  434-62. 

191 2.  "Weitere  Untersuchungen  liber  die  Befruchtung  der 
Seeigeleier  durch  Wurmsperma,"  Arch,  fiir  Zcllforsch- 
nng,  Band  8,  pp.  352-95. 

LiLLiE,  Frank  R. 

1913.  See  references  at  end  of  chapter  iv. 
LoEB,  Jacques. 

1903.  ''Uebcr  die  Befruchtung  von  Seeigeleiern  (kirch  See- 
sternsamen,"  Arch,  fiir  die  ges.  Physiol.,  Band  99, 
P-  323- 

1904.  "Further  Experiments  on  Heterogeneous  Hybridiza- 
tion in  Echinodcrms,"  Univ.  of  Cal.  Pub.,  Physiol- 
ogy, II,  15-30. 

1912.  ''Heredity  in  Heterogeneous  Hyl)rids,"  Jour,  of 
Morph.,  XXIII,  1-15. 

1914.  "Ueber  den  Mechanismus  der  heterogencn  Befruch- 
tung," Arch,  fiir  Eutwickelungsmcch.,  Band  40,  pp. 
310-21. 


2  24  PROBLEMS  OF  FERTILIZATION 

LoEB,  J.,  King,  W.  O.,  and  Moore,  A.  R. 

1910.  "Ueber  Dominanzerscheinungen  bei  den  hybriden 
Pluteen  des  Seeigels,"  Arch,  fiir  Entwickelungsmech., 
Band  29,  p.  354. 

Maupas,  E. 

1900.  "Modes  et  formes  de  reproduction  des  nematodes," 
Arch,  de  zool.  exp.  et  gen.,  Ser.  3,  T.  8,  pp.  463-624. 

MOENKHAUS,    W.   J. 

1904.  "The  Development  of  the  Hybrids  between  Fundulus 
heteroclitus  and  Menidia  notata  with  Especial  Refer- 
ence to  the  Behavior  of  the  Maternal  and  Paternal 
Chromatin,"  Am.  Jour,  of  Anai.,  Ill,  29-65. 
1910.  "Cross-Fertilization  among  Fishes,"  Proc.  of  the  Ind. 
Acad,  of  Sciences,  pp.  S53-93- 
Morgan,  T.  H. 

1904.  "Self- Fertilization  Induced  by  Artificial  Means," 
Jour.  Exp.  Zool.,  I,  135-77. 

1905.  "Some  Further  Experiments  on  Self-Fertilization  in 
Ciona,''  Biol.  Bull.,  VIII,  313-30. 

1910.  "Cross-  and  Self-Fertilization  in  Ciona  intestinalis,''^ 
Arch,  fiir  Entwickelungsmech.,  Band  30,  pp.  206-35. 

1913.  Heredity  and  Sex  (see  chap.  vii).  New  York: 
Columbia  University  Press. 

Morris,  Margaret. 

1914.  "The  Behavior  of  the  Chromatin  in  Hybrids  be- 
tween Fundulus  and  Ctenolabrus,"  Jour.  Exp.  Zool., 
XVI. 

Newman,  H.  H. 

1908.  "The  Process  of  Heredity  as  Exhibited  by  the 
Development  of  Fundulus  Hybrids,"  Jour.  Exp. 
Zool.,  V,  505-63- 

1910.  "Further  Studies  of  the  Process  of  Heredity  in  Fun- 
dulus Hybrids,"  ibid.,  VIII,  1 13-61. 

1914.  "Modes  of  Inheritance  in  Teleost  Hybrids,"  ibid., 

XVI,  447-500. 

191 5.  "Development  and  Heredity  in  Heterogenic  Teleost 
Hybrids,"  ibid.,  XVIII,  511-76. 


SPECIFICITY  IN  FERTILIZATIOX 


22 


Pi'LUGER,   E. 

1882.  "Die  Baslardzcugung  bci  den  Batrachiern,"  Arch. 
JUr  d.  ges.  Physio!.,  Band  29,  pp.  48-75. 

PFLiJGER,    E.,    AND    SmITH,    W.    J. 

1883.  '' Untcrsuchungen  uber  Bastardicrung  der  anuren 
Batrachier  und  die  Principien  der  Zeugung,"  ibid., 
Band  32,  pp.  519-80. 

PiNNEY,  Edith. 

1918.  '^\  study  of  the  Relation  of  the  Behavior  of  the 
Chromatin  to  Development  and  Heredity  in  Teleost 
Hybrids,"  Jour,  of  M  or  ph.,  XXXI,  225-90. 

Potts,  F.  A. 

1910.     "X^otes  on  the  Free-living  Nematodes,"  Quart.  Jour: 
Micr.  Sci.,  N'.S.,  LV,  433-84. 
Reynolds,  E. 

1915-  ''Prognosis  of  Sterility,"  Jour.  Amcr.  Med.  Assoc, 
LXV,  1 151-56. 

Shearer,  De  Morgan,  and  Fuchs. 

1913-  "On  the  Experimental  Hybridization  of  Echinoids," 
Phil.  Trans.  Roy.  Soc.  London,  Ser.  B.,  CCIV,  255- 
362. 

Smith,  G.  W. 

1906.  Rhizocephala— Fauna  und  Plora  des  Golfes  Neapel, 
J\Iono.  X'o.  29. 

Sobotta,  J. 

191 1.  "Ueber  das  Verhalten  der  Spermatozoen  im  Uterus 
der  Saugetiere;  nach  den  Befunden  bei  Xagetieren," 
Zcitschr.  fiir  Morph.  u.  Anthropol.,  Band  13,  pp. 
201-8. 

Stout,  A.  B. 

1916.  ''Self-  and  Cross-Pollinations  in  Cichorium  intyhus 
with  Reference  to  Sterility,"  Mem.  New  York  Bot. 
Garden,  VI,  333-454. 

1917-  Fertility  in  Cichorium  intyhus;  the  Sporadic  Occur- 
rence of  Self- Fertile  Plants  among  the  Progeny  of 
Self-Sterile  Plants,"  Amcr.  Jour.  Bot.,  l\\  375-95'. 


2  26  PROBLEMS  OF  FERTILIZATION 

Tennent,  David  H. 

1910.  ''Echinoderm  Hybridization,"  Pub.  No.  ij2,  Carne- 
gie Institution,  Washington  (other  references  to  same 
author  here). 

V^ERNON,    H.    M. 

1900.     "  Cross- Fertilization   among   the  Echinoidea,"   Arch. 
Jiir  Entwickelungsmech.,  Band  9,  p.  464. 
Waldstein  und  Eckler. 

1 9 13.     "Der  Nachweis  resorbierten  Spermas  in  weiblichen 
Organismus,"  Wiener  klin.  Wocheiischrifl,  Jahrg.  26, 
p.  1689. 
Whitman,  CO. 

1891.  "  Spermatophores  as  a  Means  of  Hypodermic  Im- 
pregnation," Jour,  of  M  or  ph.  y  \o\.  IV. 


CHAPTER  VH 
THE  PROBLEM  OF  ACTIVATION 

The  egg  may  be  activated,  caused  to  develop,  either 
by  fertilization  or  by  various  artificial  means  that 
produce  parthenogenesis.  Fertilization  involves  also 
the  factors  of  specificity  and  of  heredity,  but  experi- 
mental parthenogenesis  deals  with  activation  alone,  and 
by  virtue  of  variety  of  methods  has  become  a  most 
instructive  method  of  studying  this  problem. 

Two  phases  of  activation  are  readily  distinguished. 
In  the  first  of  these  the  plasma  membrane  and  cortex 
of  the  egg  are  affected;  in  the  second  the  internal 
protoplasm,  and  finally  the  nucleus,  are  affected,  leading 
up  to  a  karyokinetic  process,  the  first  cleavage  of  the 
egg.  The  outstanding  fact  in  activation  of  the  egg  is 
that  it  is  a  process  which  begins  in  the  cortex  and 
extends  toward  the  center.  Activation  is  usually  con- 
sidered "incomplete"  if  it  does  not  terminate  in  a 
normal  cleavage,  but  there  may  be  different  reasons 
for  such  "incompleteness." 

I.    ACTIVATION   BY   THE    SPERMATOZOON 

The  point  of  view  from  which  analysis  must  begin 
is  the  fact,  demonstrated  by  experimental  partheno- 
genesis, that  the  egg  is  an  independently  activablc 
system.  The  old  idea  that  the  spermatozoon  supi>lies 
organs  or  substances  necessary  for  activation  must  there- 
fore be  abandoned.    The  egg  possesses  all  substances 

227 


2  28  PROBLEMS  OF  FERTILIZATION 

needed  for  activation;  the  spermatozoon  is  an  inciting 
cause  of  those  reactions  within  the  egg  system  on  which 
development  depends. 

It  is  probable  that  the  spermatozoon  starts  the  acti- 
vation of  the  egg  before  entering,  and  that  penetration 
of  the  spermatozoon  into  the  egg  is  thereby  facilitated; 
penetration  of  the  spermatozoon  is  not,  as  such,  the 
cause  of  activation.  In  the  case  of  Nereis  this  is  very 
clearly  seen  because  the  spermatozoon  remains  external 
for  a  long  time  after  the  egg  has  given  numerous  other 
evidences  of  activation  (see  pp.  51-52).  Loeb  has  also 
shown  fhat  in  the  hybrid  fertilization  of  sea  urchin 
eggs  by  starfish  sperm  the  Qgg  may  in  some  instances 
exhibit  activation  by  membrane  formation  without 
entrance  of  the  sperm,  which  is  then  permanently  ex- 
cluded, as  though  the  time  for  such  form  of  reaction 
on  the  part  of  the  egg  had  passed.  But  in  most  animals 
the  act  of  inclusion  of  the  spermatozoon  within  the  egg 
is  very  rapid,  and  membrane  formation  or  other  cortical 
changes  follow  immediately. 

The  first  step  in  fertilization  is  a  more  or  less  specific 
binding  or  agglutination  of  the  spermatozoon  to  the 
egg.  We  have  seen  that  ova  of  sea  urchins  and  of 
some  other  forms  secrete  a  substance  that  produces  an 
adhesive  quality  in  spermatozoa  and  causes  them  to 
agglutinate  (pp.ii2ff.).  In  19 13  I  pointed  out  that  "the 
adhesive  property  that  the  sperm  develops  under  these 
circumstances  may  be  an  important  factor  in  binding 
the  sperm  to  the  egg  until  it  can  be  incorporated,"  and 
that  this  reaction  furnished  ''evidence  of  an  intimate 
chemical  combination  of  sperm  and  egg  constituents 
which  begins  at  the  very  moment  of  union," 


THE  PROBLEM  OF  ACTIVATION  229 

In  the  next  year  I  attempted  to  show  that  the  pres- 
ence  of   the   agglutinating   substance  is  necessary  for 
activation  in  the  sea  urchin,  basing  the  argument  upon 
three  classes  of  facts:    (i)  Fertilized  eggs,  which  are  of 
course  incapable  of  reactivation,  produce  no  more  of 
this  substance,  which  disappears  at  the  moment  of  ferti- 
lization.    (2)   Eggs  activated   by   butyric   acid,    which 
are  incapable  of  fertilization,  likewise  produce  no  more 
of  it.     (3)  If  eggs  are  subjected  to  repeated  washings 
their  production  of  this  substance  gradually  declines, 
and  along  with  it  the  capacity  for  fertilization  also. 
To  the  last  point  Loeb  has  objected  that  the  general 
vitaHty  of  the  eggs  is  also  declining  under  these  circum- 
stances;   while  this  may  be  so  in  the  case  of  the  first 
experiments  that  I  performed,  the  objection  does  not 
apply  with  equal  force  to  other  experiments  in  which 
the  protecting  jelly  of  the  eggs  was  first  removed  by 
shaking  before  beginning  the  series  of  washings;    under 
these  circumstances  the  parallel  loss  of  agglutinating 
power  and  of  capacity  for  fertilization  went  on  much 
more  rapidly. 

Moore  (19 16)  has  also  found  a  parallel  loss  of  agglu- 
tinating power  and  of  capacity  for  fertilization  in 
Arhacia  by  graded  butyric  acid  treatment  for  partheno- 
genesis. He  has  also  shown  that  eggs  exposed  to  a 
temperature  of  35°  C.  lose  their  agglutinating  power  and 
their  capacity  for  fertilization  simultaneously.  It  re- 
quires a  temperature  of  about  41°  C.  to  cause  cytolysis 
in  these  eggs,  and  the  evidence  is  that  exposure  to 
35°  C.  does  not  destroy  their  general  vitality;  they 
remain  intact  for  a  long  period  and,  if  inseminated, 
are  penetrated  by  the  spermatozoa,   which,   however, 


230  PROBLEMS  OF  FERTILIZATION 

exert  no  fertilizing  effect.  I  may  refer  here  also  to  the 
data  with  regard  to  other  animals  already  presented 
concerning  the  fertilizable  condition  of  the  ovum 
(pp.  139  ff.),  which  show  that  the  loss  of  fertilizing  power 
may  be  exceedingly  rapid  and  certainly  not  connected 
with  a  loss  of  vitality  on  the  part  of  the  eggs.  These 
data  are  most  readily  understood  on  the  assumption 
of  loss  of  agglutinating  substance  on  the  part  of  the 

egg. 

Just  (19 196)  has  found  also  in  the  case  of  Echina- 
rachniiis  that  the  production  of  the  sperm-agglutinating 
substance  is  an  index  of  the  fertihzation  capacity  of  the 
eggs;  immature  eggs  incapable  of  fertihzation  produce 
none  of  it;  ripe  eggs  washed  in  sea- water  until  they 
no  longer  give  the  agglutination  reaction  are  incapable 
of  being  fertilized,  and  during  the  washing  process  loss 
of  capacity  for  fertilization  runs  parallel  to  loss  of  agglu- 
tinating substance;  fertilized  eggs  have  lost  their  ag- 
glutinating substance,  as  have  those  with  membranes 
fully  formed  artificially.  Those  eggs  that  are  highest  in 
agglutination  capacity  fertilize  most  readily  and  produce 
the  most  vigorous  larvae. 

The  conception  that  agglutination  of  the  sperma- 
tozoon to  the  egg  is  a  necessary  factor  in  fertilization 
may  be  understood  in  one  of  two  senses:  either  that 
such  intimate  association  is  needed  for  the  further 
action  of  the  spermatozoon,  whatever  that  may  be,  or 
in  the  more  special  sense  that  the  agglutinating  sub- 
stance is  also  the  substance  that  activates  the  egg,  and 
that  it  is  set  in  operation  by  the  spermatozoon.  In 
favor  of  the  latter  more  special  interpretation  is  the 
fact  that  spermatozoa  may  enter  eggs  devoid  of  agglu- 


THE  PROBLEM  OF  ACTIVATION  231 

tinating  substance,  but  in  such  cases  the  spermatozoon 
exerts  no  fertilizing  action  whatsoever.  Thus  unripe 
eggs  of  the  sea  urchin,  which  contain  no  agglutinating 
substance,  may  be  entered  by  spermatozoa  if  high  con- 
centrations of  sperm  are  used,  but  no  change  results  in 
the  egg,  and  the  sperm  heads  remain  entirely  unchanged 
within  the  cytoplasm.  The  same  phenomenon  may  be 
observed,  as  described  by  Moore  (19 16),  in  the  case  of 
eggs  treated  for  the  optimum  length  of  time  for  pro- 
duction of  parthenogenesis  by  butyric  acid;  these  eggs 
are  devoid  of  agglutinating  substance,  but  after  removal 
of  the  membranes  they  may  be  entered  by  numerous 
spermatozoa,  which  are  perfectly  inert  in  the  egg  cyto- 
plasm; nor  do  the  eggs  react  as  would  be  expected  if 
the  sperm  carried  a  ''fertilizing  substance." 

These  facts  do  not,  however,  definitely  prove  that 
the  agglutinating  substance  is  the  activating  substance 
of  the  egg,  but  they  at  least  show  that  there  is  a  parallel 
between  absence  or  loss  of  agglutinating  substance  and 
the  capacity  of  the  egg  for  being  activated.  The  same 
results  would  be  attained  if  there  were  two  substances 
concerned,  viz.,  an  agglutinating  substance  and  an 
activating  substance,  which  were  produced  or  lost 
simultaneously  and  which  interact  in  the  process  of 
fertilization.  This  is,  as  I  understand  it,  substantial!}' 
the  position  taken  by  Miss  Woodward  in  her  recent 
study  (1918);  but  since  the  two  effects,  the  sperm- 
agglutinating  and  the  egg-activating,  appear  and  dis- 
appear together  in  these  instances,  the  writer  assumed 
that  they  may  be  regarded  as  due  to  a  single  complex 
substance,  for  which  the  name  ''fertilizin"  appeared 
appropriate. 


232  PROBLEMS  OF  FERTILIZATION 

The  writer  (1914)  used  the  phenomenon  of  inhibition 
of  fertihzation  by  blood  of  the  species  in  an  attempt 
to  approach  this  problem  a  little  more  closely.  In 
Arbacia  the  presence  of  a  certain  concentration  of  the 
perivisceral  fluid  (blood)  of  certain  individuals  inhibits 
fertilization  completely.  This  is  not  because  aggluti- 
nation is  prevented,  for  the  sperm  will  agglutinate  in 
any  concentration  of  such  blood.  Neither  is  it  merely 
a  general  colloid  effect,  such  as  Robertson  (191 26)  held 
might  inhibit  fertilization,  because  the  blood  of  some 
individuals  has  no  such  action.  The  writer  there- 
fore reasoned  that  the  action  of  the  blood  might  be 
directed  against  the  activating  substance  of  the  egg; 
if  this  were  so,  and  if  the  activating  substance  were 
contained  in  the  secretions  of  the  eggs  like  the  ag- 
glutinating substance,  it  should  then  be .  possible  to 
neutralize  the  inhibiting  action  of  the  blood  by  saturat- 
ing it  with  egg  secretions,  because  the  inhibiting  sub- 
stance would  then  be  combined.  As  a  matter  of  fact 
it  was  found  that  blood  which  is  first  treated  with  large 
quantities  of  eggs,  and  which  therefore  possesses  a  high 
agglutinating  power,  has  lost  its  power  of  inhibiting  ferti- 
lization. The  inhibiting  action  of  the  blood  may  therefore 
be  regarded  as  a  deviation  effect,  in  the  sense  that  the 
activating  substance  in  the  presence  of  blood  exerts  its 
effect  on  some  constituent  of  the  blood  and  not  on  the  egg. 
This  still  does  not  prove  that  sperm  agglutination 
and  egg  activation  are  due  to  the  action  of  a  single 
substance,  but  it  shows  again  by  a  different  method 
that  the  capacity  for  producing  both  effects  is  present 
simultaneously  in  the  egg  secretion,  and  the  assumption 
of  a  single  substance  is  the  simplest  hypothesis. 


THE  TROBLEM  OF  ACTIVATION  233 

The  conception  of  the  mechanism  of  fertihzation 
resulting  from  these  considerations  would  thus  be  that 
a  substance  borne  by  the  egg  (fertilizin)  exerts  two 
kinds  of  actions,  (i)  an  agglutinating  action  on  the 
spermatozoon  and  (2)  an  activating  action  on  the  egg. 
In  other  words  the  spermatozoon  is  conceived,  by  means 
of  a  substance  which  it  bears  and  which  enters  into 
union  with  the  fertilizin  of  the  egg,  to  release  the  activ- 
ity of  this  substance  within  the  egg. 

Without  stopping  here  to  consider  this  matter  in 
detail  let  us  note  how  the  conception  fits  the  main 
principles  of  fertilization.  In  the  first  place  it  is 
consistent  with  the  major  thesis  that  the  egg  is  an 
independently  activable  system;  whether  the  fertilizin 
is  activated  by  the  spermatozoon  or  in  some  other 
way  should  make  no  difference,  except  in  a  quantitative 
sense  in  certain  cases,  in  the  development  of  the  egg. 
In  the  second  place  it  explains  the  association  of  acti- 
vation of  the  egg  with  fertilization.  In  the  third  place 
it  explains  the  prevention  of  polyspermy,  because,  as  I 
have  shown  (p.  237),  all  free  fertilizin  is  bound  in  some 
way  at  the  moment  of  fertilization;  fertilized  eggs  pro- 
duce no  more  of  it.  The  method  of  bincHng  we  shall 
consider  later.  In  the  fourth  place  it  explains  why 
spermatozoa  are  inert  in  immature  eggs,  for  these  eggs 
have  not  yet  produced  any  fertilizin,  as  I  have  shown. 
It  is  also  entirely  consistent  with  the  facts  of  merogonic 
fertilization  as  determined  by  Delage  and  Wilson 
(p.  162).  Finally,  it  furnishes  a  basis  for  understanding 
the  problem  of  specificity,  because  the  agglutination 
phenomenon  exhibits  considerable  specificity  as  we 
have  seen,  and  it  is  in   certain  respects  analogous   to 


234  PROBLEMS  OF  FERTILIZATION 

immunity  agglutination,  in  which  the  specific  factor  is 
very  marked. 

Loeb  (1914,  1915,  1916)  has  raised  the  following 
objections  to  the  conception:  (i)  That  the  action  of 
■the  egg  secretions  on  spermatozoa  is  probably  not  a 
true  agglutination;  an  objection  that  he  later  with- 
drew. (2)  That  the  agglutinating  substance  is  derived 
not  from  the  egg  but  from  the  jelly  layer  surrounding 
the  egg.  This  also  is  incorrect,  as  I  have  shown  in  a 
separate  paper  (191 5;  cf.  also  Just,  19 19).  (3)  That 
eggs  deprived  of  their  jelly  by  acid  produce  no  more 
fertilizin  and  yet  are  capable  of  fertilization.  I  have 
shown  (Lillie,  191 5)  that  they  do  produce  fertilizin  as 
long  as  they  remain  fertilizable.  (4)  That  eggs  of  sea 
urchins  activated  by  butyric  acid  and  hence  devoid  of 
fertilizin  are  yet  fertilizable.  This  objection  has  been 
fully  discussed  already  (pp.  165-67).  (5)  That  aggluti- 
nation is  sometimes  absent  in  hybrid  fertilization;  thus 
specifically  ''the  supernatant  sea- water  of  Strongylocen- 
trotus  franciscamis  will  not  induce  cluster  formation 
[i.e.,  agglutination]  of  the  sperm  of  S.  purpiiratus;  yet 
the  latter  sperm  fertilizes  the  eggs  oi  franciscanus.^^ 

The  last  objection  requires  some  consideration. 
Agglutination  of  sperm  is  merely  an  indicator  of  the 
presence  of  a  certain  substance  which  is  none  the  less 
present  in  S.  franciscanus,  as  proved  by  agglutination 
of  its  own  sperm,  even  if  S.  purpiiratus  sperm  does  not 
reveal  it;  it  may  nevertheless  be  activated  by  S.  pur- 
pur  atus  sperm,  and  this  is  all  that  the  theory  requires. 
The  phenomenon  of  agglutination  of  sperm  with  each 
other  is  not  of  the  least  significance  as  such  in  fertiliza- 
tion, which  consists  in  the  union  of  a  single  spermato- 


THE  PROBLEM  OF  ACTIVATION  235 

zoon  with  the  egg.  It  is  a  useful  indicator  that  enables 
us  to  make  certain  analyses,  but  the  same  ])rinci])le  of 
fertilization  may  hold  in  the  entire  absence  of  sperm 
agglutination.  The  spermatozoon  is  moditied  in  the 
presence  of  egg  secretions  of  the  same  species,  by  union 
of  a  substance  in  the  spermatozoon  (agglutinable  sub- 
stance) with  a  substance  in  the  egg  secretion  (aggluti- 
nating substance  or  fertilizin).  If  this  union  renders 
the  sperm  heads  adhesive,  and  if  the  spermatozoa  are 
sufficiently  concentrated  and  motile,  they  will  aggluti- 
nate together,  otherwise  not.  But  a  degree  of  adhesive- 
ness insufficient  for  sperm  agglutination  may  be  quite 
adequate  for  agglutination  of  the  sperm  to  the  egg.  As 
pointed  out  in  chapter  vi  the  quantitative  aspect  of 
specificity  in  fertilization  requires  much  more  study, 
and  Loeb  has  given  no  quantitative  data  for  the  hybrid 
fertilization  in  question. 

Loeb  (191 5,  p.  279)  misrepresents  the  view  here 
developed  when  he  says  that  the  writer  has  called  the 
egg  an  antigen,  the  spermatozoon  a  complement,  and 
the  fertiHzin  an  amboceptor.  Such  a  view  is  not  even 
imphed  in  anything  I  have  ever  written.  What  I  did 
was  to  utihze  Ehrlich's  method  of  formulating  such  a 
three-body  reaction  for  a  pictorial  representation  of 
what  happens  in  the  fertilization  of  the  egg.  The  infer- 
ence from  the  diagram  given  (Lillie,  19141  P-  579^  01"^ 
a  strict  comparison  with  Ehrlich's  conception  of  hae- 
molysis, would  be  that  a  substance  borne  by  the  sperm 
corresponds  to  the  antigen,  the  fertilizin  to  the  ambo- 
ceptor, and  a  substance  contained  in  the  egg  corre- 
sponds to  the  complement.  But  it  was  not  suggested 
that  these  relations  were  established  by  an  immunity 


236  '   PROBLEMS  OF  FERTILIZATION 

reaction.  Only  once,  and  then  in  a  footnote  of  a  paper 
of  earlier  date  than  the  one  dealing  with  the  mechanism 
of  fertilization,  have  I  alluded  to  the  question  whether 
fertilization  may  be  regarded  as  involving  an  immunity 
reaction  (1913,  p.  563);  the  footnote  follows:  ^'In  the 
latter  case,  fertilization  itself  w^ould  have  to  be  regarded 
as  an  immunizing  process,  the  sperm  acting  as  anti- 
gen after  entrance  with  the  egg.  It  seems,  in  fact,  an 
almost  necessary  conception  of  the  general  principles 
of  immunity  phenomena  that  the  sperm  should  so  act. 
The  question  would  be,  of  course,  whether  there  is  a 
connection  between  any  antibodies  so  formed  and  the 
sperm  iso-agglutinins  produced  by  the  next  generation 
of  ova." 

Loeb  has  reversed  these  relations.  However,  I 
would  point  out  again  that  no  comparison  to  immunity 
phenomena  was  made  in  the  fertilizin  hypothesis:  an 
analogous  pictorial  method  of  representation  was 
adopted,  and  only  that. 

Another  misunderstanding  on  Loeb's  part  is  to 
regard  the  theory  as  dealing  with  the  egg  and  sperma- 
tozoon as  cells,  which,  as  he  well  says,  are  not. simple 
organic  compounds.  I  was  always  careful  to  speak  of 
''sperm  receptors"  borne  by  the  spermatozoon  and  ''egg 
receptors"  borne  by  the  egg  as  the  substances,  not 
otherwise  known,  concerned  in  the  activation  of  the 
egg,  together  with  the  fertilizin.  They  are  linked  in 
line  thus:    sperm  receptors-fertilizin-egg  receptors,  and 

not  directly,  as  sperm  receptors^    ^    1  izi  because 

^         \egg  receptors 

the  sperm  receptors  are  able  to  bind  the  fertilizin  in 

the  absence  of   the  egg  receptors  but   are    unable   to 


THE  PROBLEM  OF  ACTIVATION  237 

bind  the  egg  receptors  (as  shown  by  failure  of  activa- 
tion) in  the  absence  of  the  fertilizin. 

Let  us  return  to  the  fact  that  fertiUzed  eggs  produce 
no  more  fertilizin.  This  is  certainly  a  very  remarkable 
circumstance,  because  prior  to  fertilization  in  the  case 
of  the  sea  urchin  they  produce  it  in  such  al)undance 
as  to  charge  many  hundreds  of  times  their  own  bulk 
of  sea- water  with  easily  detectable  quantities.  Imme- 
diately after  fertilization  this  ceases,  and  the  eggs  no 
longer  react  to  spermatozoa.  Are  we  to  conceive  that 
the  eggs  excrete  it  all  in  connection  with  the  cortical 
changes  that  take  place  at  the  same  time  ?  Or  is  it  in 
some  way  combined  so  as  to  be  no  longer  active  ?  In 
favor  of  the  latter  point  of  view  is  the  fact  that  the 
internal  substance  of  the  eggs  is  capable  of  neutralizing 
the  sperm-agglutinating  action  of  fertilizin  (Lillie,  19 14). 
This  can  be  shown  by  cytolyzing  eggs  deprived  of  their 
jelly  in  distilled  water  and  thus  extracting  the  interior 
substances;  the  aqueous  extract  has  at  first  a  powerful 
agglutinating  effect,  which,  however,  disappears  entirely 
in  the  course  of  a  few  hours,  whereas  the  fertilizin  se- 
creted by  living  eggs  may  last  for  months.  Again,  if 
eggs  are  repeatedly  washed  for  about  forty-eight  hours 
until  their  production  of  fertilizin  is  very  much  reduced, 
and  are  then  shaken  to  pieces  in  the  sea- water  contain- 
ing the  fertilizin  which  they  themselves  have  secreted, 
the  fertilizin  present  before  the  shaking  is  neutralized. 
I  explained  this  by  supposing  that  eggs  contain  in  their 
interior  a  substance  capable  of  combining  with  the 
agglutinating  group  of  the  fertilizin,  but  which  is  sepa- 
rate from  it  as  long  as  the  c^g  is  inactive;  this  sub- 
stance I  called  anti-fertilizin.     I  therefore  proposed  the 


238  PROBLEMS  OF  FERTILIZATION 

hypothesis  that  fertihzation  causes  this  union  to  occur 
in  the  egg,  and  hence,  owing  to  such  binding  of  the 
agglutinating  side  chain  of  the  fertilizin,  no  sperm 
reaction  is  possible. 

It*  will  be  noted  that  no  postulate  is  made  concerning 
the  mechanism  of  action  of  fertilizin  on  the  egg.  It  is 
necessary  that  the  substance  be  present  within  the  cor- 
tex of  the  egg;  if  it  is  once  lost,  fertilizing  power  goes 
with  it.  It  operates  in  the  cortical  changes  of  fertili- 
zation, for  such  changes  are  absent  if  the  substance  be 
removed;  it  is  therefore  necessary  also,  at  least  indi- 
rectly and  possibly  directly  (see  p.  265),  for  the  internal 
changes.  The  suggestion  that  the  fertilizin  may  act 
as  a  catalyzer  is  perhaps  supported  to  a  certain  extent 
by  Richards  and  Woodward's  (191 5)  determination  of 
some  enzyme  analogies  of  this  substance.  The  sugges- 
tion is  in  any  event  a  natural  one,  as  is  shown  by  the 
rather  numerous  suggestions  in  the  literature  concern- 
ing the  connection  between  activation  and  enzyme 
action. 

Miss  Woodward  (1918)  agrees  that  fertilizin  is  ne- 
cessary for  fertilization:  eggs  of  Asterias  and  Arbacia, 
from  which  it  has  been  washed,  will  not  fertihze,  but 
if  secretion  (fertilizin)  be  added  to  such  eggs  before 
insemination  they  will  fertilize.  The  latter  observa- 
tion outruns  the  determination  of  the  present  writer 
and  obviously  constitutes  a  very  critical  point.  The 
dual  nature  of  fertilizin  is  shown  by  its  action  in  agglu- 
tinating spermatozoa  and  by  the  production  of  parthe- 
nogenesis in  eggs  of  the  same  species  when  concentrated 
on  them.  Such  autoparthenogenesis  was  first  described 
by  Glaser  (1914c).     The  writer  is  not  convinced  that 


THE  rROBLEM  OF  ACTR'ATION  239 

the  action  of  the  secretion  in  this  ])henomenon  is  spe- 
cific, but  it  obviously  deserves  attention.  This  dual 
action  of  the  egg  secretion  (fertiHzin)  is  due  in  Miss 
Woodward's  opinion  to  two  distinct  substances,  which 
may  be  obtained  separately  by  appropriate  chemical 
treatment.  The  one  of  these  is  a  sperm  agglutinin, 
the  other  is  a  parthenogenetic  agent.  The  latter  has 
fat-dissolving  properties,  and  is  assumed  to  be  a  lipase, 
for  which  the  name  ''lipolysin"  was  adopted.  It  has 
no  agglutinating  action  on  the  sperm  but  is  a  very 
efficient  parthenogenetic  agent. 

Miss  Woodward  then  proposes  a  theory  of  activa- 
tion in  the  following  terms:  ''The  resting  egg  contains 
enzymes  which  control  metabolism,  unsaturated  fatty 
acid  which  inhibits  enzyme  action,  and  lipolysin,  which 
reacts  with  the  unsaturated  fatty  acid  to  make  it 
innocuous."  Activation  is  then  caused  by  any  method 
that  increases  the  ratio  of  activating  enzymes  to  fatty 
acid,  such  as  increase  of  lipolysin,  which  destroys 
the  fatty  acid,  or  the  action  of  fat  solvents,  which 
produce  the  same  eftect  directly.  It  is  difticult  to 
see  how  the  spermatozoon  can  act  in  any  such  sense. 
The  theory  does  not  bring  together  fertilization  and 
parthenogenesis.  The  role  of  the  sperm-agglutinating 
component  of  the  egg  secretion  is  also  left  out  of 
account. 

This  author  thus  agrees  with  the  writer  with  refer- 
ence to  the  necessity  of  fertilizin  and  with  reference  to 
its  dual  capacity,  agglutinating  the  sperm  and  activa- 
ting the  egg.  She  believes,  however,  that  two  separate 
substances,  not  merely  two  separate  side  chains  of 
one  substance,  as  the  writer  supposed,  are  concerned 


240  PROBLEMS  OF  FERTILIZATION 

in  these  effects,  and  she  has  worked  out  a  theory 
of  the  activating  effect  which  is  entirely  original. 
This  theory  does  not  explain  why  the  sperm- 
agglutinating  and  the  egg-activating  properties  of  egg 
secretion  always  go  together,  as  Miss  Woodward  has 
herself  emphasized  in  various  places  in  her  paper; 
when  the  egg  ceases  to  produce  the  sperm-agglutinat- 
ing substance  it  has  lost  its  capacity  to  be  activated. 
These  two  properties  of  the  egg  secretion  hang  together 
normally;  their  separation  under  the  conditions  of 
chemical  analysis  may  possibly  denote  a  splitting  of  a 
single  substance  of  the  normal  egg. 

II.    EXPERIMENTAL   PARTHENOGENESIS 

The  determination  of  the  existence  of  a  substance 
in  the  egg  necessary  to  fertilization  obviously  does  not 
show  in  what  way  activation  results.  We  shall  there- 
fore consider  the  problem  of  activation  from  the  point 
of  view  of  experiments  in  parthenogenesis,  often  too 
hopefully  called  the  physicochemical  standpoint.  The 
books  of  Loeb  (1913)  and  Delage  and  Goldsmith  (1913) 
treat  this  subject  in  detail.  We  shall  confine  ourselves 
to  certain  outstanding  facts  and  theories  of  experimental 
parthenogenesis.  In  considering  these  it  should  be 
borne  in  mind  that,  though  the  number  of  forms  in 
which  parthenogenesis  has  been  experimentally  induced 
is  large,  in  another  considerable  number  of  forms  suc- 
cessful methods  have  not  been  found;  e.  g.,  in  the  entire 
vertebrate  phylum,  with  the  single  exception  of  the  frog. 
This  may  be  due  to  secondary  conditions  of  the  problem 
in  such  cases,  or  it  may  be  due  to  failure  to  reach  a 
correct  analysis  of  the  successful  results. 


THE  PROBLEM  OF  ACTIVATION  241 

Loeb  (19 1 6)  holds  that  the  essential  feature  in  the 
activation  of  the  egg,  whether  by  fertilization  or  parthe- 
nogenesis, is  the  change  underlying  membrane  forma- 
tion, which  he  conceives  to  be  cytolysis  of  the  superficial 
or  cortical  layer  of  the  egg.  His  reason  for  the  latter 
conclusion  is  that  "all  those  substances  and  agencies 
which  are  known  to  cause  cytolysis  or  hemolysis  will 
also  induce  membrane  formation."  They  are  Hsted  as 
follows:  (i)  fatty  acids;  (2)  saponin  or  solanin  or  bile  salts; 
(3)  lipoid  solvents,  e.g.,  benzol,  toluol,  ether,  chloroform, 
etc.;  (4)  bases;  (5)  hypertonic  or  hypotonic  solutions; 
(6)  rise  in  temperature;  (7)  certain  salts,  e.g.,  BaCh, 
SrCl2,  NaCNS;  (8)  the  blood  serum  or  cell  extracts  of 
certain  foreign  species.  Loeb  states  that  in  the  case  of 
the  sea  urchin  egg  such  agents,  used  so  as  to  restrict 
the  cytolysis  to  the  cortical  layer,  will  cause  membrane 
formation.  Rise  in  temperature  has,  however,  so  far 
been  ineffective  in  the  case  of  the  sea  urchin  egg,  though 
very  effective  in  the  starfish  and  in  Nereis.  In  the  case 
of  other  eggs  again,  none  of  these  methods  is  effective. 

In  the  sea  urchin  the  development  does  not  proceed 
to  cleavage  by  action  of  the  single  agent,  except  in  the 
case  of  hypertonic  solutions,  but  a  second  agency  is 
required  to  bring  about  further  development.  Hyper- 
tonic sea-water  is  the  second  agent  most  commonly 
employed;  this  when  used  for  the  proper  length  of 
time  insures  subsequent  normal  development.  Loeb 
therefore  states  that  the  action  of  the  first  agent  leaves 
the  egg  in  a  sickly  condition,  and  the  action  of  the 
second  agent  is  required  to  save  the  life  of  the  egg.  It 
is  a  corrective  agent  remedying  an  unavoidable  excess 
of  action  of  the  first  agent. 


242  PROBLEMS  OF  FERTILIZATION 

In  the  case  of  the  starfish,  however,  action  of  butyric 
acid  alone  is  sufficient  for  complete  development;  a 
corrective  agent  is  not  required. 

If  now  we  ask  what  is  the  nature  of  the  postulated 
cytolysis,  and  how  it  activates  the  egg,  Loeb  replies 
that  the  cytolysis  can  be  explained  by  assuming  that  a 
calcium  lipoid  compound  forms  a  continuous  layer 
under  the  surface  of  the  egg;  the  solution  of  such  a 
lipoid  layer  might  result  in  the  destruction  of  a  cortical 
emulsion.  It  then  becomes  necessary  to  assume  a  cata- 
lyzer to  explain  the  increase  in  rate  of  metaboHsm 
within  the  egg,  and  Loeb  therefore  suggests,  following 
Warburg  (1914),  that  the  cytolysis,  by  breaking  down 
the  cortical  emulsion,  releases  the  catalyzer,  assumed 
to  be  contained  in  the  cortex,  for  action  on  the  sub- 
strate. Increase  in  oxidations  results,  together  with 
certain  synthetic  processes.  Activation  of  the  egg 
therefore  comes  down  to  the  release  of  a  catalyzer; 
cytolysis  is  simply  the  means  by  which  this  end  is 
attained. 

The  above  is  the  barest  possible  sketch  of  Loeb's 
theory  and  gives  no  idea  of  the  numerous  experiments 
carried  on  year  after  year  since  the  time  of  his  original 
discovery  of  the  phenomenon  of  artificial  partheno- 
genesis in  1899.  To  get  an  idea  of  the  wealth  of  exper- 
imental data  underlying  Loeb's  analysis  the  reader 
must  refer  to  Loeb's  own  publications,  for  a  brief 
account  to  The  Organism  as  a  Whole  (G.  P.  Putnam's 
Sons,  19 1 6),  and  for  a  fuller  account  with  numerous 
references  to  original  papers  to  Artificial  Partheno- 
genesis and  Fertilization  (The  University  of  Chicago 
Press,  1913). 


THE  TROBLEM  OF  ACTIVATION  243 

The  fact  that  agents  and  conditions  capable  of 
producing  cytolysis  may  cause  membrane  formation  in 
the  sea  urchin  egg  merely  shows  that  cytolysis  may  be 
a  subsidiary  factor  in  the  activation  effect.  But  that 
cytolysis  is  a  more  fundamental  factor  is  proved,  accord- 
ing to  Loeb,  by  the  fact  that  the  eggs  exposed  so  as  to 
produce  membranes  die  by  cytolysis  later,  unless  saved 
by  a  second  process.  However,  any  activated  egg  not 
developing  normally  cytolyzes  sooner  or  later  from 
internal  causes.  Is  the  death  of  the  eggs  not  given  a 
second  treatment  due  to  cytolytic  action  of  the  agent 
or  to  some  internal  cause  resulting  from  activation  ? 

The  latter  alternative  seems  to  be  demonstrated  by 
the  cytological  examination  of  eggs  treated  according 
to  Loeb's  method,  which  has  been  made  especially  by 
Herlant    (1917;     see    also    Hindle,    1910).     The    eggs 
treated   by   butyric   acid   alone   live   for   from    twelve 
to   twenty-four   hours   before   cytolysis   begins.     What 
happens  during  all  this  time  ?     After  the  formation  of 
the  membrane  and  the  appearance  of  the  hyaline  zone 
at  the  cortex  the  cortical  changes  cease,  and  the  nucleus 
becomes  the  center  of  activity,  increasing  in  size  and 
moving  toward  the   center  of  the  egg.     The  nuclear 
membrane   then  disappears  and  a  monaster  develops 
around   the   group   of   chromosomes   formed   from   the 
egg  nucleus.     But  no  amphiaster  forms,   and   though 
the  chromosomes  divide  they  do  not  separate  in  two 
groups.     This  occurs  in  about  if  hours  at  15°  C,  accord- 
ing   to    Herlant.     At    the    same    time    the    cytoplasm 
becomes    active,    but    no    division    takes    place.     The 
chromosomes  return  to  the  condition  of  a  resting  nu- 
cleus;   a  second  monaster  then  appears.      This  process 


244  PROBLEMS  OF  FERTILIZATION 

is  repeated  at  least  four  or  five  times,  the  nucleus  all 
the  time  increasing  in  bulk.  The  phenomena  then 
become  less  regular  but  still  continue  to  be  rhythmical, 
and  no  cleavage  results. 

The  activated  egg,  thus  unable  to  divide,  spends  its 
energy  in  these  fruitless  ways  and  ultimately  breaks 
down  or  cytolyzes,  as  does  any  sufficiently  abnormally 
directed  egg. 

How  does  the  second  treatment  with  h>T>ertonic 
sea- water  save  the  Hfe  of  the  egg  ?  Briefly,  it  does  this 
by  giving  the  egg  the  capacity  for  regular  division  and 
thus  directing  the  energies  of  the  egg  into  normal  chan- 
nels. Morgan  (1896,  1900)  and  Wilson  (1901),  among 
others,  had  long  ago  noted  that  unfertilized  eggs  of 
sea  urchins  react  to  hypertonic  sea-water  by  forming 
asters  apart  from  the  nucleus.  Herlant  has  shown  that 
one  of  these  cytoplasmic  asters,  together  with  the  mon- 
aster associated  with  the  egg  nucleus,  form  an  amphi- 
aster  which  initiates  regular  division  of  the  egg  nucleus. 
From  this  time  on  everything  moves  normally. 

It  is  therefore  clear  that  the  changes  underlying 
membrane  formation  do  not  involve  progressive  cytol- 
ysis;  it  is  the  processes  of  activation  aroused  by  the 
cortical  changes  that  are  responsible  for  the  final  death 
of  the  egg  in  the  absence  of  proper  co-ordination  of 
nuclear  and  cell  division.  It  is  therefore  very  doubtful 
that  the  changes  underlying  membrane  formation  itself 
should  be  regarded  as  cytolytic,  unless  by  extension  of 
the  usual  meaning  of  the  term  "cytolysis."  The  nature 
of  the  cortical  changes  underlying  membrane  forma- 
tion seem  to  the  writer  still  to  be  obscure,  but  it  is 
inadvisable  to  use  a  term  with  a  purely  pathological 


THE  PROBLEM  OF  ACTIVATION  245 

connotation  for  a  process  that  occurs  in  normal  ferti- 
lization. 

Loeb  also  postulates  a  similar  mechanism  for  ferti- 
hzation.  To  make  the  parallelism  between  fertilization 
and  parthenogenesis  complete,  Loeb  emphasizes  the  fact 
that  the  sperm  may  cause  membrane  formation  with- 
out entering  the  egg,  but  that,  as  in  the  case  of  artificial 
membrane  formation  alone,  development  goes  no  far- 
ther. Thus  fertilization  exhibits  two  phases  which 
Loeb  compares  to  the  cytolytic  action  and  the  correct- 
ive action  in  his  method  of  artificial  parthenogenesis. 
It  is  true  that  any  genetic  process  admits  of  division, 
and  we  have  seen  in  discussing  partial  fertilization  that 
the  action  of  the  spermatozoon  may  be  stopped,  not 
only  at  the  moment  of  penetration,  but  at  any  time 
thereafter.  Such  a  parallelism  between  parthenogenesis 
and  fertilization  would  hold  for  any  theory  of  activation. 

Loeb  also  holds  that  the  spermatozoon  carries  a 
substance  (lysin)  into  the  egg  which  effects  an  altera- 
tion of  its  surface  layer,  apparently  of  the  nature  of  a 
cytolysis  (1916,  p.  iro).  If  this  were  so,  it  would  follow 
that  two  or  more  spermatozoa  would  increase  the  corti- 
cal changes  above  the  normal,  but  this  is  not  the  case; 
from  which  it  follows  that  the  cortical  changes  result 
from  action  of  the  egg  itself.  With  reference  to  the 
cortical  changes,  as  with  reference  to  the  later  phases 
of  fertilization,  the  spermatozoon  is  merely  an  activator, 
and  this  is  why  under  optimum  conditions  the  egg  does 
not  respond  more  energetically  to  an  excess  of  sperm 
than  to  a  single  one. 

Attempts  to  isolate  a  cytolytic  substance  from  sper- 
matozoa have  signally  failed,  as  we  have  seen  (p.  133). 


246  PROBLEMS  OF  FERTILIZATION 

in  their  purpose  of  securing  a  substance  that  will  act 
on  eggs  of  the  same  species.  But  on  the  other  hand 
watery  extracts  of  sperm  may  be  highly  cytolytic  to 
ova  of  other  species,  especially  of  different  classes  or 
phyla;  there  is,  however,  no  tissue  specificity  in  this, 
for  blood  or  tissue  exudates  may  have  the  same  effect. 
It  is  therefore  obvious  that  we  must  accept  the  nega- 
tive result  within  the  species  as  showing  that  cytol- 
ysins  borne  by  the  sperm  have  no  cytolytic  function  in 
normal  fertilization;   they  act  only  on  foreign  species. 

Loeb's  theory  is  based  mainly  on  the  study  of  the 
sea  urchin.  However  firm  may  be  our  conviction  that 
the  fundamental  phenomena  of  activation  must  be  the 
same  throughout  the  animal  kingdom,  yet  we  must  not 
fail  to  reahze  that  each  species  will  show  its  own  pecul- 
iar aspects.  In  the  case  of  the  sea  urchin  one  of  these 
is  the  sharp  separation  between  the  two  stages  on  which 
Loeb  lays  so  much  emphasis.  In  most  other  forms, 
e.g.,  starfish,  annehds,  frog,  the  activation  process 
appears  continuous,  though  capable  of  arrest  at  vari- 
ous stages.  In  the  case  of  the  frog,  however,  Herlant 
(19 13)  has  shown  that  a  separation  of  two  phases 
similar  in  principle  to  that  of  the  sea  urchin  may 
readily  be  recognized. 

R.  S.  Lillie  (1908,  1915)  has  examined  certain 
quantitative  aspects  of  activation  with  much  greater 
precision  in  the  case  of  the  starfish,  and  from  such 
determinations  different  points  of  view  naturally  arise. 
He  has  determined  that  definitely  timed  exposures  to 
supranormal  temperatures  constitute  an  almost  perfect 
method  for  producing  parthenogenesis  in  the  starfish. 
This  is  the  simplest  possible  method  of  studying  the 


THE  PROBLEM  OF  ACTIVATION  247 

subject,  for  it  is  not  complicated  by  the  presence  of 
any  unusual  substance.  The  action  may  be  varied  as  to 
both  degree  of  temperature  and  period  of  action.  A 
third  variable  factor  is  the  age  of  the  eggs  after  placing 
in  sea-water;  the  only  necessary  statement  in  the  last 
connection  is  that  the  eggs  of  the  starfish  carry  out 
their  entire  maturation  in  sea-water,  and  that  action 
of  increased  temperature  before  the  germinal  vesicle 
has  broken  down  is  not  only  ineffective  but  actually 
detrimental  to  the  subsequent  viability  of  the  eggs. 
The  condition  of  the  eggs  then  gradually  improves  for 
about  an  hour  at  the  ordinary  temperature  of  the  sea- 
water  until  the  time  of  formation  of  the  first  polar 
body,  and  from  this  optimum  point  the  eggs  deterio- 
rate. The  curve  of  condition  for  parthenogenesis  coin- 
cides  with  the  curve  for  fertilization,  as  Delage  (1901a) 
first  showed. 

The  method  of  experiment  is  to  expose  eggs  in  their 
optimum  condition  to  the  temperature  to  be  tested  by 
transferring  to  sea-water  at  the  desired  temperature, 
and  then  to  transfer  samples  back  to  sea-water  at 
normal  temperature  (i9°-2i°  C.)  at  stated  intervals. 
The  eggs  undergo  no  visible  changes  in  the  heated 
sea- water,  but  react  when  transferred  to  the  normal 
temperature.  The  following  tables  (p.  248)  showing  the 
results  at  31°  C.  and  32°  C.  will  suffice  for  the  discussion. 

The  first  table  shows  three  separate  experiments  at 
31°  C.  and  two  determinations  for  each  experiment: 
the  percentage  of  eggs  forming  membranes  and  the 
percentage  developing  to  larvae;  the  second  table  shows 
six  experiments  at  32°  C.  with  only  the  latter  determi- 
nation.    The  following  points  should  be  noted:   (t)  That 


248 


PROBLEMS  OF  FERTILIZATION 


the  percentage  of  eggs  developing   to  larvae   may  be 
as  high  by  heat  parthenogenesis  as  by  fertilization,  for 


31°  C.     THREE  EXPERIMENTS 


Duration 
OF  Expo- 
•      SURE  IN 

Minutes 


1-2 
2^ 


3h 

4- 

5- 
6. 

8. 

10 

12 

14-15 
17-18 
20-21 
25-30 


Percentages  of  Eggs  Forming  Fertilization-Membranes  and  Larvae 


Experiment   i 

Membranes 

Larvae 

0 

0 

0 

0 

Few  (<  i) 

0 

10-15 

0      . 

Ca.      20 

0 

80-90 

Ca.  2-  3 

Ca.      90 

15-20 

Ca.      90 

40-50 

70-80 

40-50 

Ca.      15 

Ca.     40 

15-20 

Ca.       5 

Experiment  2 


Experiment  3 


Membranes 


Ca. 


Ca. 
> 

> 

Ca. 


o 
o 

10-15 
30-40 

70-80 

90 
90 

QO 

100 


Larvae  I   Membranes 


Ca. 

Ca. 

> 

Ca. 
Ca.  1 1  Ca. 
20-30!  Ca. 

Ca. 


o 

5 

50 
60-70 

95 
100 

100 

100 

100 

Ca.        90 

Ca.  75-80 

40-50 

Ca.        20 


Larvae 


O 

o 
o 
o 
o 
o 
I 
20 

60 

80-90 

50-60 

Ca.  10-15 
o 


Ca. 
Ca. 
Ca. 


32°  C.     SIX  EXPERIMENTS 


Dura- 
tion of 

Expo- 
sure in 
Minutes 


1-3 
4-  ■ 

S-- 
6.. 

7-. 
8.. 
10. 
12. 

IS- 


Percentages  of  Eggs  Forming  Free-Swimming  Larvae 


Exp.  I 


o 
Ca.     I 
Ca.      3-4 
Ca.  35-40 

>         90 

85-90 


Exp.  2 


Exp.  3 


<  I 

2-  3 

20-30 

70-80 

Ca.    95 

50-55 
15-20 

o 


o 
Ca.  4-5 
15-20 
55-60 
95 
75-85 
25-35 
<i 


Ca. 


Exp.  4 

Exp.  5 

0 

0 

2-  3 

Ca.      5 

25-35 

Ca.    50 

60-70 

80-90 

>    90 

Ca.    60 

>    90 

25-35 

50-60 

<        5 

Exp.  6 

O 

o 

10-15 

25-35 
50-60 

80-90 

80-90 

Ca.    20 


it  is  rare  to  secure  80-90  per  cent  developing  larvae 
by  fertilization  with  this  material.  (2)  A  short  expo- 
sure may  cause  membrane  formation  with  no  subse- 


THE  PROBLEM  OF  ACTIVATION 


249 


quent  development  (see  the  first  table  above).  (3)  In 
each  experiment  the  percentage  of  reacting  eggs  in- 
creases with  time  of  exposure  to  an  optimum  and  then 
decreases.  (4)  The  optimum  time  for  production  of 
larvae  at  31°  is  14  to  15  minutes;  at  32°,  from  6  to  8 
minutes;  that  is,  the  rate  of  the  activation  reaction  is 
approximately  doubled  by  a  rise  of  1°  in  temperature. 
At  33°  the  optimum  exposure  is  about  4§  to  5I  minutes; 
at  34°,  3  to  4  minutes;  at  35°,  ij  to  2  minutes;  and  at 
36°,  I  to  ij  minutes. 

The  range  of  effective  temperatures  is  from  about 
29°  to  38°.  The  following  table  shows  the  time  varia- 
tions for  various  effects  within  this  range: 


APPROXIMATE  TIMES  OF  EXPOSURE  REQUIRED  TO  PRO- 
DUCE THE  FOLLOWING  EFFECTS  AT  DIFFERENT 

TEMPERATURES 


Tempera- 
ture 


29° 
30° 
31° 
32° 
33° 
34° 
35° 
36° 
37° 
38° 


Formation  of 
Membranes 


Ca.12-14  m. 
8-10  m. 
Ca.  4  m. 
Ca.  2  m. 
Ca.         I  m. 


Ca. 
Ca. 


^  to  I  m. 
^  m. 
15  sec. 


Minimum  for 
Larvae 


20-25  rn. 
Ca.   18  m. 
Ca.     8    m. 
4-5    m. 

2^-3    m. 
Ca.     2    m. 
i-ij  m. 
30-45  sec. 


Optimum  for 
Larvae 


30-40  m. 
Ca.       28  m. 
Ca.       15  m. 
7-  8  m. 
4I-52  m. 
3-3h  ni. 
1^-2^  m. 
1-15  m. 
30-35  sec. 
Ca.       20  sec. 


Maximum  for 
Larvae 


?>      30  m. 

21-25  m. 

10-12  m. 

8-10  m. 

Ca.        5  m. 

Ca.        3  m. 

Ca.        2  m. 


Commenting  upon  the  process  underlying  the  time 
variation  at  the  different  temperatures,  R.  S.  LiUie  says: 

In  endeavoring  to  form  some  consistent  conception  of  the 
nature  of  this  process,  the  following  facts  have  to  be  consid- 
ered. It  exhibits  a  high  temperature  coefhcient:  from  fifteen 
to  twenty  times  the  duration  of  exposure  is  required  to  induce 


2  50  PROBLEMS  OF  FERTILIZATION 

the  membrane-formation  at  30°  as  at  35°;  the  ratios  between 
29°  and  34°  and  between  31°  and  36°  are  the  same.  At  each 
temperature  the  proportionate  durations  of  the  minimum, 
optimum,  and  maximum  exposures  for  forming  larvae  are  approx- 
imately the  same.  In  other  words  the  critical  change  under- 
lying simple  membrane-formation  is  affected  by  temperature  in 
the  same  way  as  that  underlying  complete  activation  of  develop- 
ment: i.e.,  the  proportionate  increase  in  velocity  by  rise  of  tem- 
perature is  the  same  in  both  cases,  a  fact  which  can  only  indicate 
that  one  fundamental  process — and  not  two — is  concerned  in 
producing  both  effects.  If  we  assume  that  the  foregoing  propor- 
tionate increase  in  velocity  prevails  through  a  rise  of  10°,  a  Qio 
value  of  from  225  to  400  is  indicated,  as  against  the  2  to  3 
characteristic  of  chemical  reactions  in  homogeneous  media. 
Activation  by  heat  thus  depends  on  some  critical  change  in  the 
egg  which  does  not  begin  until  a  temperature  of  about  29°  is 
reached,  but  which  undergoes  very  rapid  acceleration  with  further 
rise  of  temperature.  The  liquefaction  of  gels  by  heat  seems  to 
be  the  only  relevant  process  which  shows  these  characteristics. 
The  change  in  viscosity  preceding  the  gelation  of  a  gelatine  sol 
undergoes  very  rapid  acceleration  with  lowering  of  temperature, 
within  a  few  degrees  of  the  temperature  of  gelation.  The  inverse 
process,  melting  of  gels,  has  a  similarly  high  temperature  coeffi- 
cient. In  general  the  facts  suggest  that  the  direct  effect  of  the 
high  temperature  is  to  cause  a  change  in  the  colloidal  system  of 
the  egg,  of  such  a  kind  as  to  render  possible  a  chemical  inter- 
action between  substances  which  in  the  normal  condition  of  the 
resting  egg  are  kept  apart.  This  restraining  condition  may  be 
some  physical  barrier  like  a  membrane,  impermeable  to  the  dif- 
fusion of  the  substances  concerned,  or  it  may  be  a  certain  state 
of  electrical  polarization  of  the  general  cell-surface. 

Two  other  important  facts  in  the  experiments  of 
this  author  should  also  be  stated:  (i)  It  is  possible 
to  arrest  the  progress  of  the  activation  process  by 
returning  the  eggs  to  sea-water  at  normal  temperature, 
and  to  cause  a  renewal  later  by  restoring  the  tempera- 


THE  PROBLEM  OF  ACTUATION  251 

ture,  and  this  without  interfering  with  the  effect. 
(2)  Precisely  similar  quantitative  relations  obtain  when 
the  same  kind  of  eggs  is  activated  by  butyric  acid  at 
normal  temperatures,  the  variables  being  concentra- 
tion and  time  in  this  case;  and  moreover  it  is  possible 
to  complete  an  incomplete  action  of  either  temperature 
or  butyric  acid  by  action  of  the  other.  ^J^hcre  can  thus 
be  no  doubt  that  the  processes  of  activation  are  the 
same  by  these  two  methods. 

R.  S.  Lillie's  conclusion  is  that  the  fundamental 
"releasing"  process  in  activation  of  the  egg  cannot 
possibly  depend  on  simple  acceleration  of  some  chemi- 
cal process,  such  as  oxidation,  for  the  temperature 
coefficient  of  such  processes  is  of  the  order  Qio  =  2-3, 
instead  of  200-400.  There  is  some  change  in  the  nature 
of  degelation  or  decrease  of  viscosity  in  the  cortical 
layer  which  presumably  allows  substances  to  come 
together  and  interact  which  in  the  condition  of  the 
cortex  of  the  unfertilized  egg  are  kept  apart.  'The 
extent  of  the  following  development  will  depend  on 
the  degree  of  completion  of  this  process.  This  is  of 
course  consistent  with  the  idea  of  various  authors  that 
the  cortical  changes  release  a  specific  catalytic  action; 
but  R.  S.  Lillie's  measurements  of  the  releasing  change 
give  us  a  much  better  idea  of  these  changes  than  we 
have  previously  had. 

Other  theories  of  activation. — The  veteran  French 
experimental  zoologist  Yves  Delage  (1908  and  1913; 
earlier  references  here)  has  propounded  a  theory  based 
upon  the  conception  that  the  phenomena  of  cell  <li\isi()n 
depend  on  a  reversible  series  of  gelations  and  degela- 
tions  in  the  protoplasm,  changes  from  gel  to  sol  and 


252  PROBLEMS  OF  FERTILIZATION 

vice  versa,  which  he  proposes  to  call  the  theory  of 
colloidal  morphogenesis.  There  is  no  doubt,  as  we  have 
previously  seen  (p.  153),  that  such  changes  are  involved 
in  cell  division  and  hence  in  the  phenomena  of  activa- 
tion that  initiate  the  first  division  ^of  the  egg.  But  it 
seems  to  the  writer  that  in  the  series  of  events  of  activa- 
tion these  changes  do  not  come  first — that  they  are 
effects  rather  than  causes  of  activation.  Delage  is  so 
conscientious  a  critic  of  his  own  theory,  which  he  says 
attributes  an  undemonstrated  role  to  intrinsic  forces 
of  the  egg,  viz.,  of  determining  other  phenomena  than 
those  directly  dependent  on  the  action  of  the  reagents, 
that  any  other  critic  must  be  disarmed.  The  concep- 
tion is,  however,  of  great  historical  interest  at  least, 
and  it  led  Delage  to  discover  one  of  the  best  methods 
yet  devised  for  activation  of  sea  urchin  eggs.  A  short 
account  of  it  is  therefore  desirable. 

Delage  regards  the  *' first  event"  (membrane  forma- 
tion), which  precedes  the  segmentation  of  the  egg,  as 
a  coagulation;  ''the  second  event"  (disappearance  of 
the  nuclear  membrane)  as  a  liquefaction.  He  thus 
came  to  the  conclusion  that  if  a  coagulative  effect  could 
be  produced  in  the  unfertilized  egg  by  one  reagent, 
and  if  this  were  followed  by  a  liquefactive  effect  pro- 
duced by  another  reagent,  development  of  the  egg 
might  be  induced.  Regarding  acids  in  general  as  coagu- 
lating agents  and  alkalies  as  liquefying  agents,  he  was 
led  to  try  the  effect  of  successive  action  of  acids  and 
alkalies  on  unfertihzed  eggs  of  the  sea  urchin.  The 
application  of  this  method  to  eggs  in  sea-water  was 
without  success,  but  it  yielded  very  beautiful  results 
in  hypertonic  sea-water.     Tannin  was  finally  selected 


THE  TROBLEM  OF  ACTRATlOX 

as  the  best  acid  reagent,  as  it  appeared  to  possess  a 
particularly  strong  coagulative  effect;  ammonia  was  used 
as  alkah.     He  still  found,   however,   that   the  method 
did  not  succeed  in  sea-water,  and  finally,  for  reasons 
that   need   not   be   considered,    adopted   a   mixture   of 
70  per  cent  sea-water  and  30  per  cent  of  a  solution  of 
saccharose  estimated  to  have  the  same  osmotic  pres- 
sure as  the  sea-water.     To  50  c.c.  of  this  mixture  he 
added  twenty-eight  drops  of  a  '^decinormal"  solution 
of  tannin;    the  eggs  were  placed  in  and  left  live  to  six 
minutes;  then  thirty  drops  of  AVio  solution  of  ammonia 
were  added  to  neutralize  the  acid  and  to  render  the 
mixture  slightly  alkaline.     After  an  hour  the  eggs  were 
washed  and  allowed  to  develop  in  sea-water.     Delage 
states  that  as  many  eggs  may  develop  by  this  method 
as  by  fertilization,   and   he   even   raised   some   larvae 
through  metamorphosis. 

The  method  is  thus  very  complex   and   inevitably 
suggests  the  question  whether  it  acts  according  to  the 
theory  of  causing  a  coagulation  followed  by  a  lique- 
faction.    Miss    Lloyd    (1914),    working    under    Loeb's 
direction,  has  made  an  analysis  of  this  method.     She 
points  out  that  the  sugar  solution  employed  was  strongly 
hypertonic   as   compared   with   sea-water   and   showed 
that  the  tannic  acid  is  not  necessary.     The  tannic  acid 
employed  by  Delage  was  really  a  1/60  molecular  solution, 
according  to  later  views  of  its  molecular  structure,  instead 
of  ^'decinormal,"  as  Delage  supposed,  and   hence  the 
ammonia  added  renders  the  solution  strongly  alkaline. 
The  activation  is  therefore  attributed  by  this  author  to 
the  hypertonic  action  of  sugar  solution  combined  with 
the  strong  alkahne  reaction  of  the  medium,  both  of  which 


254  PROBLEMS  OF  FERTILIZATION 

Loeb  has  shown  to  be  effective  activating  factors  in 
the  sea  urchin. 

Authors  who  have  attributed  a  directly  activating 
effect  to  changes  in  the  physical  state  of  the  colloids 
of  the  egg  are  Fischer  and  Ostwald  (1905)  and  Heil- 
brunn  (1915);  the  latter  author's  results  have  been 
reviewed  previously  (p.  00).  The  view  that  he  main- 
tains is  "that  the  only  physico-chemical  effect  which 
all  parthenogenetic  agents  possess  in  common  is  the  pro- 
duction of  a  gelatinization  (or  coagulation)  within  the 
egg.  Hence  I  regard  this  gelatinization  (or  coagulation) 
as  the  direct  cause  of  the  initiation  of  development" 
(1915,  p.  191).  In  what  sense,  however,  coagulation  may 
be  considered  to  activate  the  egg  is  by  no  means  clear; 
the  only  demonstrable  connection  is  between  coagulation 
and  cell  division,  but  the  coagulation  involved  there  is 
strictly  localized  (p.  000)  and  not  general.  For  the  pro- 
cess of  membrane  formation  itself  Fleilbrunn  (19 13)  has 
a  different  explanation,  viz.,  that  it  is  produced  by  a 
lowering  of  the  surface  tension  of  a  pre-existing  mem- 
brane, which  is  then  pushed  away  from  the  egg  by  the 
internal  forces  which  previously  balanced  its  greater 
tension.  It  is  only  by  regarding  membrane  formation 
as  a  mere  epiphenomenon  that  the  subsequent  coagu- 
lation can  be  treated  as  a  primary  activating  factor. 
However,  the  phenomenon  of  the  primary  cortical  change 
is  too  general  to  be  treated  in  this  fashion,  and  its 
character  in  different  animal  groups  is  too  varied  for  it 
to  be  a  mere  phenomenon  of  decrease  of  surface  tension. 
The  egg  activated  by  a  spermatozoon  also  coagulates  in 
the  same  sense,  as  Heilbrunn  has  shown  by  his  measure- 
ments, but  Heilbrunn  does  not  maintain  that  the  sperma- 


THE  PROBLEM  OF  ACTIVATION 


255 


tozoon  introduces  a  coagulating  agent;    the  coagulation 
IS  not  the  cause  but  a  result  of  activation. 

It  must  already  be  obvious  that  it  is  difticult  to 
settle  the  question  of  priority  of  incidence  of  the  physi- 
cal and  chemical  changes  involved  in  the  activation  of 
the  egg.     A  second  difficulty  equally  great  is  to  show- 
how  these  changes  intervene  in  the  physiological  events 
of  activation,  and  a  third  is  to  show  how  the  morpho- 
logical sequence  results  as  a  consequence  of  physico- 
chemical    and    physiological    events.     But    these    are 
absolutely  general  biological  problems,  and  the  subject 
of  activation  of  the  egg  is  probably  as  far  advanced 
with  respect  to  them  as  any  other  biological  problem, 
in  some  respects  more  so.     Investigation  will  naturally 
follow  these   three  directions  more  or  less  separately 
and  simultaneously.     But  a  view  that  does  not  respect 
all  three  fields  will  necessarily  be  partial  and  incomplete. 
The  discussion  of  parthenogenetic  activation  would 
be  too  incomplete  if  it  did  not  include  an  account  of 
Bataillon's  brilliant  success  (1910)  in  producing  parthe- 
nogenesis in  the  frog,  and  of  the  analysis  of  the  results 
by  Bataillon  and  other  investigators.     As  is  well  known, 
this  worker  after  years  of  vain  attempts  to  induce  the 
development  of  frogs'  eggs  by  parthenogenesis  llnally 
succeeded  by  the  exceedingly  simple  methofl  of  pricking 
them  with  a  fine  needle.     This  result  has  been  conlirmed 
by  Dehorne  (1911),  Henneguy  (191 1),  Brachet  (191 1), 
Loeb    and    Bancroft    (1913),   McClendon    (1912),   and 
Herlant  (1913,  191 7).     As  a  matter  of  historical  justice 
it  should  be  mentioned  that  this  result  was  foreshadowerl 
by  Guyer  (1907)  in  his  remarkable  experiments  of  inject- 
ing blood  into  frogs'  eggs,  by  which  some  devcloi)ment 


256  PROBLEMS  OF  FERTILIZATION 

was  initiated.  In  a  careful  analysis  of  his  own  results 
Bataillon  showed  that  for  complete  activation  by 
pricking  it  was  necessary  that  blood  or  tissue  extract 
should  be  carried  into  the  egg  by  the  needle;  otherwise 
the  development  was  arrested  without  cleavage.  Herlant 
•  (1913,  191 7)  has  confirmed  this  and  has  furnished  a 
simple  cytological  explanation  of  the  result:  Eggs  that 
are  pressed  from  the  body  of  the  uninjured  frog  and 
pricked  with  a  clean  needle  complete  maturation  and 
form  a  small  first  segmentation  spindle  so  near  the 
center  of  the  egg  that  its  division  produces  no  eftect  on 
the  cytoplasm  of  the  egg,  which  remains  undivided. 
But  if  the  eggs  be  moistened  by  blood  before  pricking, 
asters  arise  in  the  cytoplasm  at  the  point  of  pricking, 
and  by  extension  so  affect  the  first  segmentation  spindle 
that  it  remains  relatively  near  the  surface  of  the  egg. 
The  division  of  this  spindle  then  involves  that  of  the 
egg  cytoplasm  also,  and  development  proceeds  normally. 
Tadpoles  obtained  by  pricking  have  been  reared  to 
maturity  by  Loeb  and  Bancroft. 

This  method  points  to  some  very  simple  physical 
change  of  the  cortex  as  the  primary  event  in  activation. 
Bataillon  believed  that  the  cortical  change,  which  he 
spoke  of  as  a  contraction  of  the  egg,  involved  an  excre- 
tion of  fluid  containing  substances  such  as  CO,  that 
inhibited  development. 

It  is  significant  that  recent  studies  in  parthenoge- 
netic  activation  (Loeb,  R.  S.  Lillie,  Bataillon)  point  to 
changes  in  the  cortical  zone  of  the  egg  protoplasm  as 
the  primary  factor  in  activation.  While  the  precise 
nature  of  these  changes  still  remains  obscure,  it  seems 
obvious  that  their  effect  is  to  release  reactions  within 


THE  PROBLEM  OF  ACTIVATION'  257 

the  cortical  zone  that  were  previously  inhibited  (Loeb, 
R.  S.  Lillie).  It  is  natural  then  to  associate  the  rising 
rate  of  metaboHsm  with  catalytic  action,  as  Loeb  has 
done.  But  it  should  be  borne  in  mind  that  this  is 
hypothetical,  for  such  a  reaction  has  not  been  demon- 
strated. It  is  then  supposed  that,  when  these  reactions 
have  been  released,  the  complex  mechanism  of  the  egg 
is  set  in  action;  but  if  the  initial  reaction  is  quanti- 
tatively incomplete,  or  otherwise  imperfect,  the  mechan- 
ism stops  sooner  or  later,  owing  in  some  cases  perhaps 
to  too  slow  a  rate,  in  other  cases  certainly  to  lack  of 
co-ordination.  Such  conditions  are  in  some  cases  sus- 
ceptible of  adjustment  by  secondary  treatments,  as  we 
have  seen. 

III.    DISCUSSION 

I.  The  cortical  changes. — Eggs  that  are  artificially 
activated  always  exhibit  a  marked  slowness  in  rate  of 
development,  even  with  the  best  methods,  as  compared 
with  fertilized  eggs.  This  suggests  some  factor  in  ferti- 
lization that  has  not  yet  been  successfully  imitated  iii 
any  artificial  way.  But  apart  from  this  the  activation 
theories  resulting  from  parthenogenesis  are  not  directly 
apphcable  to  the  action  of  the  spermatozoon.  The 
manner  of  activation  must,  however,  be  the  same  for 
the  most  part;  comparison  of  fertilization  and  parthe- 
nogenesis should  therefore  serve  to  complete  the  theory 
of  activation.  The  most  sigm'ficant  factor  that  fertili- 
zation has  to  add  to  the  theory  of  acti\'ation  would 
appear  to  be  the  association  of  capacity  for  fertilization 
(activation)  with  a  diffusible  substance  or  substances 
(fertilizin)  contained  in  the  cortex  of  the  egg.  The 
theory  of  artificial  activation  requires  the  presence  of 


258  PROBLEMS  OF  FERTILIZATION 

catalytic  substances  for  the  interactions  that  follow  the 
primary  cortical  effect;  but  their  nature  has  remained 
entirely  unknown. 

Just's  experiments  (1915^)  on  heat  parthenogenesis 
in  Nereis  furnish  an  experimental  basis  for  the  view 
that  the  activating  substance  is  readily  diffusible.  He 
found  that  the  eggs  could  be  stimulated  to  complete 
parthenogenetic  development  by  proper  heat  exposure, 
provided  that  they  were  not  first  washed  in  sea-water. 
If  they  were  first  washed,  the  eggs  lost  their  capacity 
for  complete  heat  activation,  although  they  were  still 
fertilizable.  This  difference  can  be  readily  understood 
on  the  assumption  that  the  spermatozoon  is  a  more 
efficient  activator  than  heat,  and  that  there  remains 
after  washing  a  sufficient  amount  of  activable  substance 
for  action  of  the  spermatozoon,  but  not  of  heat.  We 
have  previously  pointed  out  that  the  sea-water  used 
for  washing  the  eggs  contains  a  sperm-agglutinating 
substance,  and  we  have  given  the  reasons  for  identifying 
this  with  the  activating  substance  (pp.  229  ff.). 

How  can  the  spermatozoon  exert  an  effect  on  the 
cortex  similar  to  that  produced  by  heat  or  butyric 
acid?  Heilbrunn  (191 5)  suggests  that  the  spermato- 
zoon produces  a  partial  liquefaction  or  swelling  of  the 
vitelline  membrane  at  the  point  of  attachment,  thus 
lowering  the  surface  tension  at  one  point;  there  is  an 
immediate  tendency  for  the  tension  to  be  equalized 
everywhere,  which  results  in  a  certain  lowering  of  the 
tension  of  the  membrane  around  the  entire  egg,  and 
elevation  of  the  membrane  (usually  called  membrane 
formation)  results,  owing  to  an  overbalance  of  forces 
acting  outward  on  the  membrane.     Such  an  explana- 


THE  PROBLEM  OF  ACTUAllON  259 

tion  might  perhaps  suffice  in  a  formal  sense  for  the 
special  case  under  consideration  by  Heilhrunn,  that  of 
the  sea  urchin.  But  it  can  hardly  apply  to  other  cases 
where  the  cortical  changes  present  a  different  morpho- 
logical form  {Nereis,  for  instance,  F'ig.  2).  Loeb  has 
suggested  that  the  sperm  bears  a  lysin  which  acts  on 
the  surface  of  the  egg;  the  objections  to  this  concep- 
tion have  been  already  considered  (p.  245),  and  it 
seems  to  the  writer  untenable.  R.  S.  Lillie  (1914)  has 
suggested  the  idea  that  contact  of  the  sperm  produces 
an  electrical  depolarization  of  the  membrane  which 
releases  an  ''impediment  to  the  chemical  interaction 
forming  the  primary  event  in  the  response''  (p.  614). 

There  are  good  reasons  for  believing  that  the  propa- 
gation of  the  cortical  change  from  the  point  of  impact 
of  the  spermatozoon  requires  a  measurable  time  ele- 
ment. It  has  been  asserted  by  several  observers,  begin- 
ning with  Fol  (1877),  that  the  fertilization  membrane 
can  be  seen  to  arise  first  at  the  point  of  penetration  and 
to  spread  thence  as  fast  as  the  eye  can  follow  it;  this 
is  a  very  delicate  observation,  owing  to  the  difficulty 
of  seeing  the  precise  point  of  fertilization  at  the  moment 
of  impact.  It  is  therefore  difficult  to  say  how  much 
maybe  due  to  subjective  impressions  in  this  observation. 

Just  (19 1 9)  has,  however,  recently  observed  a  case 
in  the  sand  dollar  {Echinarachniiis),  where  the  rate  of 
the  cortical  change  can  be  readily  followed  by  the  eye; 
membrane  elevation  can  be  observed  to  j^roceed  as  a 
wave  around  the  egg,  beginning  at  the  point  of  i)ene- 
tration  of  the  sperm.  The  sperm  enters  the  egg  in 
about  twenty  to  fifty  seconds  after  insemination;  the 
membrane    begins    to    arise   at    the    point   of  entrance 


26o  PROBLEMS  OF  FERTILIZATION 

about-  fifteen  seconds  later,  it  and  is  completed  in 
fifteen  to  twenty  seconds  more.  In  this  case  the  entire 
egg  has  already  become  impermeable  to  sperm  at  the 
moment  that  membrane  elevation  has  begun.  There 
is  in  fact  a  ''wave  of  negativity"  that  sweeps  over  the 
egg  preceding  the  wave  of  membrane  elevation,  so  that 
any  point  on  the  surface  becomes  impermeable  to  sperm 
(unfertilizable)  some  time  before  membrane  elevation 
begins.  The  wave  of  negativity  dates  from  the  time 
that  the  tip  of  the  sperm  head  has  entered  the  cyto- 
plasm. The  so-called  fertilization  cone  arises  after 
penetration  of  the  spermatozoon. 

The  principles  of  polyspermic  fertilization  seem  to 
the  writer,  also,  to  furnish  a  demonstration  of  the  point 
in  question.  With  a  perfectly  fresh  lot  of  eggs  one 
can  raise  the  percentage  of  polyspermy  by  increasing 
the  concentration  of  the  sperm  suspension.  Since  the 
egg  becomes  unfertilizable  at  any  point  where  the  ferti- 
lization reaction  has  begun,  we  would  expect  that  poly- 
spermy would  increase  with  sperm  concentration  if  there 
were  an  appreciable  time  interval  in  the  spread  of  the 
reaction  from  a  point,  and  this  is  what  we  observe. 
With  a  given  sperm  con.centration  also  the  proportion 
of  polyspermic  eggs  increases  with  weakening  of  the 
eggs  either  by  action  of  reagents  or  by  staling.  The 
obvious  implication  here  is  that  in  weakened  eggs 
the  cortical  change  is  propagated  more  slowly.  It  is 
noteworthy  in  this  connection  that  normal  polyspermy 
occurs  only  in  eggs  with  considerable  yolk  content  and 
hence  large  surface. 

The  conclusion  that  the  cortical  change  is  propa- 
gated from  the  point  of  fertilization  at  a  rapid  but 


THE  PROBLEISI  OF  ACTIVATION  2O1 

measurable  rate,  which,  however,  \'aries  with  tlie  kind 
of  eggs  and  with  their  condition,  is  well  founded.  Such 
a  change  is  not  comparable  to  electrical  depolarization 
or  decrease  of  surface  tension.  The  rate  at  which  the 
cortical  reaction  spreads  .from  the  point  of  action  of 
the  spermatozoon  furnishes  a  new  basis  of  judgment 
concerning  the  probable  nature  of  the  change.  The 
general  order  of  magnitude  is  of  a  physiological  rather 
than  a  purely  physical  sort.  It  is  perhaps  hardly  neces- 
sary to  say  that  the  activation  of  the  egg  at  a  gi\en 
point  by  the  spermatozoon  does  not  present  a  different 
problem  from  artificial  activation.  It  does,  however, 
give  an  additional  point  of  view  with  reference  to  it. 

The  primary  change  in  activation  is  not  something 
visible  in  a  morphological  sense;  the  visible  cortical 
changes  are  due  to  activation  and  are  obviously  specific 
for  the  kind  of  egg  concerned;  hence  the' considerable 
variety  that  they  exhibit  in  different  animal  groups. 
Some  theories  of  artificial  activation  have  erred  in 
respect  to  undue  emphasis  on  the  morphological  change 
as  though  it  were  primary  instead  of  being  secondary 
or  even  tertiary.  The  analysis  implies,  first,  a  physical 
alteration  at  the  point  of  fertilization;  second,  chemical 
reaction  in  which  a  specific  catalyzer  is  concerned;  third, 
the  visible  cortical  eft'ect.  The  writer  is  not  full\-  con- 
vinced that  the  chemical  reactions  may  not  be  set  up 
directly,  but  this  point  is  perhaps  a  minor  one. 

Loeb  (1910)  has  suggested  that  in  the  process  of 
cytolysis  which  he  conceives  to  underly  the  activation 
of  the  egg  "certain  substances  which  were  solid  are 
liqufied  and  enabled  to  diffuse  into  the  egg.  If  it 
could  be  shown  that  these  substances  were  of  such  a 


262  PROBLEMS  OF  FERTILIZATION 

nature  as  to  start  or  accelerate  the  chemical  processes 
underlying  development  the  connection  between  mem- 
brane formation  and  causation  of  development  would 
become  intelligible."  This  idea  seems  to  me  to  be 
entirely  consistent  with  the  fertilizin  hypothesis.  This 
substance,  which  is  contained  in  the  cortex,  may  be 
conceived  as  exerting  a  ferment-hke  action  as  it  pene- 
trates into  the  egg  or  is  carried  in  by  the  spermatozoon, 
though  this  conception  must  remain  for  the  present 
hypothetical.  It  corresponds  to  what  I  have  called 
elsewhere  (1914)  binding  of  the  ovophile  group  of  the 
fertilizin. 

2.  The  internal  changes. — The  problem  of  the  in- 
ternal events  of  activation  presents  two  aspects:  first, 
that  of  change  in  metabolism  evidenced  by  increased 
oxygen  consumption  in  some  cases,  by  initiation  of 
development  in  all  cases;  and  second,  the  problem  of 
proper  co-ordination  of  the  karyokinesis  of  the  first 
cleavage.  Under  the  first  head  it  would  seem  that  all 
degrees  of  activation  are  possible  up  to  the  optimum, 
which  in  this  case  appears  to  correspond  with  the  max- 
imum, for  we  do  not  know  any  cases  in  which  defects 
in  the  induced  development  are  due  to  excessive  acti- 
vation, i.e.,  to  too  rapid  rate  of  the  processes  activated 
or  initiated.  As  we  have  seen,  polyspermy  does  not 
induce  such  a  condition;  its  evil  effects  result  from 
other  causes;  neither  does  excessive  action  of  partheno- 
genetic  agents  cause  an  excessive  increase  in  rate  of 
metabolic  activities,  but  rather  a  decreased  rate.  It  is 
possible  to  grade  the  action  of  parthenogenetic  agents 
to  various  degrees  of  activation;  beginning,  for  in- 
stance,  with   the  production  of  membrane  formation 


THE  PROBLEM  OF  ACTIVATION  263 

alone  as  the  first  rcadil)-  observed  evidence  of  activa- 
tion, a  slight  increase  in  action  of  the  agent  ina\-  bring 
eggs  to  the  point  of  cleavage;  slightly  more  may  induce 
cleavage  in  a  small  proportion  of  the  eggs;  more  yet  a 
larger  proportion  of  normally  developing  eggs,  up  to 
the  optimum  action,  beyond  which  the  reaction  becomes 
unfavorable  (cf.  R.  S.  Lillie,  191 5). 

Under  the  second  head  it  is  not  only  necessary  that 
the  rate  and  kind  of  metabolism  should  reach  a  certain 
optimum,  but  also  that  the  processes  initiated  should 
be  properly^  co-ordinated.  Thus  if  the  processes  within 
the  egg  exhibit  a  lack  of  co-ordination  with  reference 
to  regular  segmentation,  the  entire  developmental  pro- 
cess may  go  astray,  whatever  its  rate.  This  is  often 
the  case  with  artificial  activation,  as  we  have  seen  in 
discussing  the  necessity  for  a  double  treatment  in 
parthenogenesis  of  the  sea  urchin  (p.  243). 

In  the  case  of  fertilization,  the  karyokinetic  phe- 
nomena center  around  the  sperm  nucleus  at  first,  and 
the  egg  nucleus  is  later  drawn  into  the  same  sphere  of 
influence.  We  have  already  discussed  the  theory  that 
the  leading  part  taken  by  the  sperm  component  is  due 
to  a  special  organ  (centrosome)  intimately  associated 
with  it  (p.  71).  There  is  no  good  reason  for  adhering 
to  this  view,  which  has  been  quite  generally  aban- 
doned. 

We  have  seen  that  spermatozoa  that  penetrate  into 
immature  eggs,  into  eggs  devoid  of  agglutinating  sub- 
stance, or  into  eggs  already  activated  exert  no  effect 
in  the  interior.  As  the  spermatozoon  j^roduces  no  cor- 
tical effect  in  these  cases,  we  might  generalize  by  saying 
that  a  spermatozoon  that  has  not  been  concerned  in 


264  '  PROBLEMS  OF  FERTILIZATION 

cortical  reactions  cannot  enter  into  the  internal  reac- 
tions of  fertilization.  This  might  be  due  either  to  a 
change  in  the  spermatozoon  itself  during  its  passage 
through  the  cortex,  to  a  change  in  the  internal  proto- 
plasm of  the  egg  consequent  on  the  cortical  changes, 
or  to  both  combined.  It  would  be  difficult  to  separate 
these  possibilities,  because  we  cannot  isolate  a  spermato- 
zoon that  has  been  concerned  in  cortical  activation 
and  introduce  it  into  central  protoplasm  (endoplasm) 
of  another  unactivated  egg. 

However,  a  very  ingenious  experiment  of  Chambers' 
throws  some  hght  on  the  problem.  It  has  been  shown 
by  other  experimenters  (see  p.  162)  that  portions  of 
fertilizable  eggs  are  themselves  fertilizable;  such  parts 
possess  a  portion  of  the  cortex  (ectoplasm)  of  the  egg. 
Chambers  has  added  a  most  interesting  and  significant 
fact  by  showing  that  the  internal  protoplasm  (endo- 
plasm) of  the  starfish  egg  is  not  fertilizable.  With  the 
microdissection  needle  he  tore  the  cortex  and  allowed 
endoplasm  to  flow  out;  this  accumulates  in  spheres, 
which  may  be  of  considerable  size  and  may  contain 
the  egg  nucleus.  Of  forty  to  fifty  such  endoplasmic 
spheres  not  one  could  be  fertilized;  on  the  other  hand 
the  cortical  material  left  behind,  which  rounds  up  into 
a  sphere  in  each  case,  is  fertilizable.  If  an  endoplasmic 
mass  which  has  flowed  out  of  a  tear  be  allowed  to 
remain  connected  with  cortical  material  the  mass  is 
fertilizable,  and  the  regularity  with  which  segmentation 
of  such  a  mass  proceeds  is  a  function  of  the  amount 

^The  writer  is  greatly  indebted  to  Dr.  Chambers  for  permission  to 
record  these  observations  in  advance  of  his  own  publication  on  the 
subject. 


THE  PROBLEJM  OF  ACTIVATION  265 

of  cortical  material  present.  With  a  minimum  (,f  cor- 
tical material  a  fertilization  reaction  occurs  and  nuclear 
division  may  follow,  but  in  such  a  case  the  cytoplasm 
does  not  divide. 

The  writer  (1914)  has  previously  urged  the  possi- 
bility that  the  spermatozoon  undergoes  some  modifica- 
tion, necessary  for  its  part  in  the  internal  events  of 
fertilization,  in  its  passage  through  the  cortex  of  the 
tgg,  possibly  by  union  with  fertilizin  or  other  substance 
of  the  cortex;  in  other  words,  that  -the  spermatozoon 
needs  itself  to  be  fertilized." 

The  material  of  the  sperm  nucleus  is  in  a  different 
ph3'siological  state  from  the  egg  nucleus.     In  the  cases 
m  which  the  sperm  enters  the  egg  during  maturation 
the  egg  nucleus  is  concerned  in  the  maturation  divisions- 
m  those  cases  (e.g.,  sea  urchins)  in  which  maturation 
is  completed  before  penetration  of   the  spermatozoon 
the  egg  nucleus  is  in  the  resting,  interkinetic,  vesicular 
state,  while  the  sperm  nucleus  is  in  the  state  of  greatest 
condensation  of  the  chromatin;    but  in  either  ^ase  the 
sperm  nucleus  is  associated  with  cytoplasm  which  has 
received  the  cortical  activation  and  which  max-  hence 
be  regarded  as  more  reactive.     It  is  therefore 'natural 
that  the  karyokinetic  phenomena,  which  constitute  the 
normal  reaction  of  the  egg,  should  center  around  the 
sperm  nucleus,  and  the  formation  of  the  sperm  aster 
may  be  regarded  as  the  first  step  in  this  process.     The 
cytological  study  of  artificial  activation  has  shown  that 
the  cytoplasm  has  a  tendency  when  activated  to  form 
asters    spontaneously    (Morgan,    Wilson).     When    the 
sperm    nucleus    is    present    all    such    activit\-    centers 
around  it  and  is  usually  inhibited  elsewhere. 


266  PROBLEMS  OF  FERTILIZATION 

The  karyokinetic  phenomena  thus  initiated  tend  to 
occupy  the  entire  egg,  but  they  are  in  conflict  with  the 
maturation  divisions  when  these  have  not  aheady 
occurred,  and  their  extension  is  in  such  cases  delayed. 
But  after  this  conflict  has  disappeared  the  extension  is 
very  rapid.  The  meeting  of  the  germ  nuclei,  as  previ- 
ously argued  (p.  65),  is  due  to  their  relation  to  the 
single  dynamic  system  of  the  entire  egg,  in  which  they 
tend  toward  the  center  according  to  the  universal  rule 
governing  nucleocytoplasmic  localization.  There  is  no 
reason  for  assuming  any  such  vague,  semimystical  con- 
ception as  a  '' sexual  affinity  "  of  the  germ  nuclei. 

Many  of  the  phenomena  concerned  in  the  internal 
events  of  fertilization  or  artificial  activation  are  of  a 
general  cytological  kind;  the  writer  has  therefore  not 
seen  fit  to  discuss  them,  as  this  is  not  a  treatise  on  gen- 
eral cytology.  We  have  attempted  to  isolate  from  the 
complex  of  events  those  which  are  peculiar  to  fertili- 
zation as  such  and  to  indicate  their  relations  to  known 
physiological  processes. 

3.  General. — As  will  have  been  seen  from  the  preced- 
ing discussion  most  of  the  theories  of  fertilization  are 
activation  theories  alone.  Boveri's  theory  of  the  cen- 
trosome,  though  entirely  morphological,  is  such  a 
theory;  such  also  are  the  various  theories  of  Loeb, 
R.  S.  Lillie,  Delage,  and  others  who  have  used  arti- 
ficial parthenogenesis  as  a  means  of  analysis.  On  the 
other  hand,  certain  older  theories,  such  as  that  of  Oskar 
Hertwig,  take  cognizance  mainly  of  the  problem  of 
biparental  inheritance  presented  by  fertilization.  There 
is  obviously  need  for  a  theory  that  shall  comprise  the 
main  fundamental  features  of  the  fertilization  reaction, 


THE  PROBLEIM  OF  ACTIVATION  267 

viz.,  specificity,  irreversibility,  and  activation,  for  these 
are  inseparable.  The  penetration  of  the  spermatozoon 
and  the  union  of  the  germ  nuclei  are  results  of  these 
primary  factors  of  the  fertilization  reaction,  and  they 
constitute,  therefore,  secondary  problems,  even  though 
from  a  teleological  point  of  view  they  represent  the 
essence  of  the  entire  process. 

It  is  hoped  that  the  writer's  fertilizin  hypothesis 
presented  in  various  places  in  this  book  at  least  supple- 
ments other  theories  of  activation  and  points  the  way 
to  a  more  inclusive  theory  that  shall  comprise  all  the 
main  aspects  of  fertilization. 

REFERENCES 

Allyn,  Harriet  M. 

191 2.     "The  Initiation  of  Development   in   Chaetoptcrus," 
Biol.  Bull.,  XXIV^,  21-72. 

Bataillon,  E. 

1909.     "L'impregnation  heterogene  sans  amphimixie  nucle- 

aire  chez  les-amphibiens  et  les  echinodermes,"  Arch. 

fur  Entwickehaigsmech.,  T,  38. 
i9iO(?.     "L'embryogenese  complete  provoquee  chez  les  am- 

phibiens  par  piqure  de  I'oeuf  vierge,  larves  parthc- 

nogenetiques   de    Rami   fusca,^'   Com  pics  rcndus    de 

I'Acad.  de.  Sci.,  T.  150. 
1910/;.     Le    probleme    de    la    fecondation    circonscrit    par 

I'impregnation  sans  amphimixie  et  la  parthenogenese 

traumatique,"  .4rc//.  de  zool.  exp.  et  gen.,  Ser.  5,  T.  6, 

pp.  101-35. 
1911a.     "Les  deux  facteurs  de  la  parthenogenese  trauma- 

tiques    chez    les    amphibiens,"    Com  pics    rcndus    dc 

VAcad.  de  Sci.,  T.  152. 
1911/'.     "L'embryogenese  provoquee  chez  I'a'uf  vierge  d'am- 

phibicns  par  innoculation  dc  sang  ou  de  spcrmes  de 

mammifercs,"  etc.,  ihid. 


268  PROBLEMS  OF  FERTILIZATION 

Bataillon,  E. 

191 2.  "La  parthenogenese  des  amphibiens  et  la  'feconda- 
tion  chimique'  de  Loeb  (etude  analytique),"  Ami. 
des.  sci.  nat.,  Ser.  9,  T.  16,  pp.  249-307. 

Bracket,  A. 

191 1.  "Etudes  sur  les  localisations  germinales  et  leur  poten- 
tialite  reelle  dans  I'oeuf  parthenogenetique  de  Rana 
fusca,"  Arch,  de  bioL,  T.  26,  pp.  337-63. 

Dehorne,  a. 

191 1.  "Sur  le  nombre  des  chromosomes  dans  les  larves 
parthenogenetiques  de  grenouille,"  Comptes  rendiis  de 
VAcad.  de  Sci.,  T.  152,  p.  11 23. 

Delage,  Y. 

1901a.     See  references  at  end  of  chapter  v. 

1902.  "Nouvelles  recherches  sur  la  parthenogenese  experi- 
mentale  chez  Asterias  glacialis,^'  Arch,  de  zool.  Exp. 
et  gen.,  Ser.  3,  T.  10,  pp.  213-35. 

1904a.  "Elevage  des  larves  parthenogenetiques  d' Asterias 
glacialis,''  ibid.,  Ser.  4,  T.  2,  pp.  27-46. 

1904&.  "La  parthenogenese  par  I'acidal  carbonique  obtenu 
chez  les  ceufs  apres  Remission  des  globules  polaires," 
ibid.,  pp.  43-46. 

1908.  "Les  vrais  facteurs  de  la  parthenogenese  experi- 
mental. Elevage  des  larves  parthenogenetiques 
jusque  a  forme  parfaite,"  ibid.,  Ser.  4,  T.  7,  445-508. 

Delage,  Y,  et  Goldsmith,  M. 

1913.  La  parthenogenese naturelle  et  experimentale.  Paris: 
Ernest  Flammarion. 

Fischer,  Martin  H.,  and  Ostwald,  Wolfgang. 

1905.  "Zur  physicalisch-chemischen  Theorie  der  Befruch- 
tung,"  Arch.  JUr  d.  ges.  Physiol.,  Band  106,  pp.  229- 
66. 

GUYER,  M.   F. 

1907.  "The  Development  of  Unfertilized  Frogs'  Eggs  In- 
jected with  Blood,"  Science,  N.  S.,  XXV,  910-11. 


THE  PROBLEM  OF  ACTIVATION  269 

Heilbrunn,  L.  V. 

1913.     ';Studies  in  Artincial  Parthenogenesis:    I,  Membrane 
Lleva  ion    in    the    Sea-Urchin    Kgg."    Biol     Bull 
A-^AV,  343-61. 
191 5.     See  references  at  end  of  chapter  v. 
PIenneguy,  M.  F. 

1911.     ''Sur  laparthenogenese  experimentale  chez  les  amphi- 
biens,     Comptes  rendu s  de  VAcad.  de  ScL,  T    1.2   nn 
121-23.  '  ^' 

Herlant,  Maurice. 

1917.     ';Le  mechanisme   de   la   parthenogenese  experimen- 
tale,    Bull.  sa.  de  la  France  el  de  la  Belgique   Ser   7 
^-  50,  pp.  381-404.  ■  '' 

1913.  j'Etude  sur  les  bases  cytologiques  du  mecanisme  de 
la  parthenogenese  experimentale  chez  les  amphi- 
bians,   Arch,  de  biol,  T.  2^,  pp.  505-60S. 

HiNDLE,    E. 

1910.     ^\  Cytological  Study  of  Artificial  Parthenogenesis 
m  btrongylocentrolus  purpuratus^  Arch,  fur  Entwick- 
elungsmech.,  Ba.nd  SI. 
Just,  E.  E. 

191 5*.     See  references  at  end  of  chapter  v. 
LiLLiE,  Frank  R. 

1 9 13.  See  references  at  end  of  chapter  iv. 

1914.  See  references  at  end  of  chapter  iv. 
191 5«.     See  references  at  end  of  chapter  iv. 
1915^-     See  references  at  end  of  chapter  iv. 

LiLUE,  R.   S. 

190S.     "Momentary  Elevation  of  Temperature  as  a  Means 
of  Producing  Artificial   Parthenogenesis   in   Starfish 
Eggs  and  the  Conditions  of  Its  Action,"  Jour   Exp 
Zool.,  V,  375-428. 
1910.     "The  Physiology  of  Cell-Division:    II,  The  Action 
of  Isotonic  Solutions  of  Neutral  Salts  on  Unfertilized 
Eggs  of  Asterias  and  Arhaciar  Am.  Jour,  of  Phx.siol., 
XX\T,  106-33. 


270  PROBLEMS  OF  FERTILIZATION 

LiLLIE,    R.    S. 

191 1.  "Certain  Means  by  Which  Starfish  Eggs  Naturally 
Resistant  to  Fertilization  May  Be  Rendered  Normal 
and  the  Physiological  Conditions  of  This  Action," 
Biol.  Bull,  XXII,  328-46. 

1913a.  "The  Role  of  Membranes  in  Cell-Processes,"  Pop. 
Sci.  Monthly,  pp.  132-52. 

19136.  "The  Physiology  of  Cell-Division:  V,  Substitution 
of  Anaesthetics  for  Hypertonic  Sea-water  and  Cy- 
anide in  Artificial  Parthenogenesis  in  Starfish  Eggs," 
Jour.  Exp.  ZooL,  Vol.  XV. 

1914.  "Antagonism  between  Salts  and  Anaesthetics:  IV, 
Inactivation  of  Salt  Solutions  and  Hypertonic  Sea- 
water  by  Anaesthetics,"  Jour.  Exp.  ZooL,  XVI, 
591-616. 

1916.  "Mass  Action  in  the  Activation  of  Unfertilized  Star- 
fish Eggs  by  Butyric  Acid,"  Jour,  of  Biol.  Chemistry, 
XXIV,  233-47. 

191 5.  See  references  at  end  of  chapter  v. 

1916.  See  references  at  end  of  chapter  v. 

191 7.  See  references  at  end  of  chapter  v. 

1918.  See  references  at  end  of  chapter  v. 

Lloyd,  Dorothy  J. 

1914.  "A  Critical  Analysis  of  Delage's  Method  of  Produ- 
cing Artificial  Parthenogenesis  in  the  Eggs  of  the  Sea- 
Urchin,"  Arch,  filr  Entivickelungsmech.,  Band,  38,  pp. 
402-8. 

LoEB,  Jacques. 

1910.  "How  Can  the  Process  Underlying  ^Membrane  For- 
mation Cause  the  Development  of  the  Egg?"  Proc. 
Soc.  Exp.  Biol,  and  Medicine,  VII,  120-21. 

1913.  Artificial  Parthenogenesis  and  Fertilization.  Chicago: 
The  University  of  Chicago  Press. 

1914.  See  references  at  end  of  chapter  iv. 

191 5.  See  references  at  end  of  chapter  iv. 

1916.  See  references  at  end  of  chapter  iv. 


THE  PROBLEM  OF  ACTI\^\T10N  271 

LoEB,  Jacques,  and  Bancroft,  F.  W. 

"The  Sex  of  a  Parthcnogcnctic  Tadpole  and  Frog," 
Jour.  Exp.  Zool.,  XIV,  275-77. 

McClendon,  J.  J. 

191 2,  ''Dynamics  of  Cell  Division.  Artificial  Partheno- 
genesis in  Vertebrates,"  Am.  Jour,  of  Physiol., 
XXIX,  298-301. 

Moore,  Carl  R. 

1916.  See  references  at  end  of  chapter  v. 

191 7.  See  references  at  end  of  chapter  v. 

Morgan,  T.  H. 

1896.  "The  Production  of  Artificial  Astrospheres,"  Arch. 
fiir  Entwickelungsmcch.,  Band  3,  pp.  339-61. 

1900.  "Further  Studies  on  the  Action  of  Salt  Solutions 
and  of  Other  Agents  on  the  Egg  of  Arbacia,''  Arch, 
fiir  Entwickelungsmcch. ,  Band  10,  pp.  489-524. 

Richards,  A.,  and  Woodward,  A.  E. 

191 5.     See  references  at  end  of  chapter  iv. 

Robertson,  T.  B. 

191 2&.     See  references  at  end  of  chapter  v. 

Wilson,  E.  B. 

1901.  "Experimental  Studies  in  Cytology:  I,  A  Cytological 
Study  of  Artificial  Parthenogenesis  in  Sea-Urchin 
Eggs,"  Arch,  fiir  Entwickelungsmech.,  Band  12,  pp. 
529-96. 

Woodward,  Alvalyn  E. 

1918.  "Studies  on  the  Physiological  Significance  of  Certain 
Precipitates  from  the  Egg  Secretions  of  Arbacia  and 
Asterias,^^  Jour.  Exp.  Zool.,  XXVT,  459-502. 


INDEX 


INDEX 


Activable  substance  of  ovum,  167, 
171,  258  (sec  also  Fertilizin) 

Activation,  problem  of,  228  ff. 

Agglutinating  substance,  117  ff., 
120,  154,  167,  211,  228  ff.,  234' 
235  (see  also  under  Spermatozoa, 
agglutination  of) 

Alkali,  effect  on  fertilization,  24, 
170771,  191,  193 

Amphimixis,  32,  36,  38 

Antagonism  of  sperm,  a  76 

Anti-fertilizin,  237 

Arhacia,  100,  109,  m,  112,  117, 
118,    122,    123,    124,    147,    151,' 


154,  164,  174 
Aristotle,  i,  2,  3 
Ascaris,  59,  66,  69,  75,  147 

■  i'^'T''^}\^'  "•^'  '70, 174,  193 

Auerbach,  L.,  14 
Auto-parthenogenesis,  238 

Balance  of  salts  in   fertilization 

172,  194 
Ballowitz,  E.,  49,  93 
Balzer,  F.,  189,  191,  193 
Bancroft,  F.  W.,  255,  256 
Barry,  12 

Bataillon,  E.,  203,  255,  256 
Berlese,  185 

Biparental  inheritance,  47 
Bischoff,  T.  L.  W.,  10,  II 
Blood  inhibition,  27,  173  ff    2^2 
Blount,  Mary,  79     '    ^^    ''    ^ 
Bonnevie,  K.,  -jS,  80 
Boveri,  T.,  19,  60,  66,  71,  74,  ^2, 

162,  266 
Brachet,  A.,  84,  225 
Braun,  204 

S''i^^'i:r^-^^-'93-98,  114 
Butschh,  O.,  14,  2>7 

Butyric  acid  treatment  in  parthe- 
nogenesis, 157,  166,  229,  241, 
242,  251 


Calkms   G  N.,  39,  40,  42 
Castle,  W.  E.,  205  ff. 


Catalysers,  242 
Chaetoptcnis,  46,  loi,  140,  176 
Chambers,  R.,  153,  154,  ^54 
Chemotaxis,  50,  102  ff.,  114 
Child,  C.  M.,  34 
Chromosome  reduction,  45 

Chromosomes  in  fertilization    66- 
67 

Cohn,   E.   J.,   92,    100,    loi,   I03, 
132  ■^' 

Colloidal   morphogenesis,   Theory 

of,  252 
Colton,  H.  S.,  204 
Conjugation,  37  ff. 
Conklin,  E.  G.,  72 
Cook,  A.  H.,  204 
Correns,  C,  205,  212,  213  ff. 
CO2  production  at  fertiHzation,  147 
Cytolysis,  241  ff.,  261 

Darwin,  C,  205,  211,  215 

Dehorne,  A.,  255 

Delage,  Y.,  25,  140,  143,  162,  233, 

240,  251  ff.,  256 
De  Meyer,  J.,  114,  120 
De  Morgan,  188,  193 
Dewitz,  J.,  93,  98 
Doncaster,  L.,  188 
Driesch,  H.,  162 
Dumas  (Prevost  et),  9,  10,  it 
Dungay,  N.  S.,   160 

East,  E.  M.,205,  212,  215,  216 

Echinarachnius,  113,  117,  119,  124, 
151,  230 

Eckler,  185 

Egg:  extracts,  109,  no;  nucleus, 
16,  63,  158;  receptors,  27,  236; 
secretions,  loS,  109,  no 

Ehrlich,  235 

Electrical  conductivity,  148 

Endomixis,  39,  40 

Erdmann,  R.,  39 

External    conditions    of    fertiliza- 
tion, 170  ff. 


27s 


276 


PROBLEIMS  OF  FERTILIZATION 


Fertilizable    condition    of    ovum, 

139  ff.,  163 
Fertilization  cone,  51,  55 
Fertilization  reaction,  22,  23,  130 
Fertilizin,  27,  140,  142,  175,  231, 

.233,  237,  238,  267 
First  segmentation  nucleus,  65 
Fischel,  A.,  190 
Fischer,  M.  H.,  254 
Flemming,  17 
Fol,  H.,  15,  16,  58,  161 
Fuchs,  H.  M.,  132,  188,  193,  205, 
207  ff. 

Gametes,  31 

Gelation  phenomena,  153,  254 

Gemmill,  J.  J.,  138 

Germ  nuclei,  60,  65,  68,  158 

Gies,  W.  J.,  133 

Glaser,  Otto,  116,  118 

Godlewski,  E.,  169,  176,  195 

Goldsmith,  M.,  240 

Gray,  J.,  100,  148,  188,  189 

Grobben,  49 

Giinther,  G.,  100,  199 

Guyer,  M.  F.,  255 

Harper,  E.  H.,  78,  79 

Hartsoeker,  N.,  5 

Harvey,  E.  N.,  149 

Harvey,  William,  i,  2,  3 

Heat  parthenogenesis,  143,  246  ff., 
258 

Heilbrunn,L.V.,  153,  254,  258,  259 

Henking,  H.,  60,  78 

Henneguy,  M.  F.,  255 

Herbst,  Curt,  169,  189,  193 

Herlant,  M.,  81,  83,  176,  177,  243, 
246,255,  256 

Hertwig,  G.,  202,  203 

Hertwig,  O.,  15,  16,  21,  23,  25,  81, 
140,  160  ff.,  256 

Hertwig,  P.,  199 

Hertwig,  R.,  21,  23,  81,  140,  162 

Hetero-agglutination,  122  ff. 

Hindle,  E.,  243 

Hybrid  fertihzation,  186  ff.;  in 
echinoderms,  188  ff.;  in  tele- 
osts,  i98ff.;  in  amphibia,  200  ff. 

Hypertonic  sea- water,  in  parthe- 
nogenesis, 164,  241,  244 


Inbreeding,  215 

Inhibition  of  fertilization  by  blood, 

173  ff.,  232 
Iso-agglutination,  122,  123,  218 

Jennings,  H.  S.,  39  ff. 
Jost,  L.,  205,  213,  215 
Just,  E.  E.,  25,  85,  117,  119,  124, 

140,  141,  144,  154,  236,  234,  258, 

259 

Keber,  12 

Kohlbrugge,  J.  H.  F.,  184,  185 
Kolliker,  A.,  10,  11,  12,  100 
Kostanecki,  K.,  56 
Kupelwieser,  H.,  196,  197 

Lallemand,  11,  17 

Lams,  75 

Leeuwenhoek,  A.,  4,  5 

Lillie,  R.  S.,  24,  143,  149,  150,  151, 
160,  246,  249,  251,  256,  259,  263, 
266 

Lipolysin,  239 

Lloyd,  D.  J.,  253 

Loeb,  J.,  23,  28,  58,  95,  104,  III, 
113,  116,  118,  124,  132,  142,  145, 
148,  156,  159,  164,  169,  171,  177, 
187,  191, 193,  228,  234ff.,  24off., 
245,  254  ff.,  261,  266 

Lyon,  E.  P.,  147,  148,  149 

Lysin,  132,  177,  245 

McClendon,  J.  J.,  148,  255 

McGregor,  J.,  49 

Mall,  F.  P.,  139 

Mark,  E.  L.,  18 

Massart,  J.,  93,  98 

Maturation,  44,  45 

Maturity  of  gametes,  24 

Maupas,  E.,  37,  38,  39,  40,  204 

Meissner,  G.,  12 

Merogony,  68,  161  ff. 

Meves,  F.,  60,  71,  75 

Minot,  C.  S.,  32 

Mitochrondria,  20,  50,  70,  75,  76 

Moenkhaus,  W.  J.,  198,  199,  200 

Moore,  C.  R.,  25,  118,  154,  155, 

165,  167  ff.,  229,  231 
Morgan,  T.  H.,  162,  204,  205,  207, 

208,  209,  210,  215,  244,  265 


INDEX 


277 


Morrill,  C.  V.,  68 
Morris,  M.,  199 
Mrazek,  56 
Mulsow,  K.,  68,  6q 

Nereis,  46,  51,  52,  62,  69,  72,  76, 
94,  99,  104,  105,  113,  122,  123, 

139,  153.  154,  15s,  159,  258 
Newman,  H.  H.,  198  ff. 
Newport,  G.,  12,  84 

Okkelberg,  P.,  148 
Osmotic  relations,  149  ff. 
Ostwald,  W.,  254 
Oxidation  changes,  145  ff. 

Paramecium,  37  ff. 
Parthenogenesis,   8,    21,    22,    130, 

143,  159,  163  ff.,  240  ff. 
Partial  fertilization,    129,    155  ft'.. 

165 
Patius,  5 

Permeability  change,  147  ff. 
Pfliiger,  E.,  201 
Physical  changes,  152  ff. 
Pinney,  E.,  199 
Polar  bodies,  44 
Polyspermy,  51,  77,  78,  79,  80,  81, 

82,  83,  161,  260,  262 
Potts,  F.  A.,  204 
Prevost  et  Dumas,  9  ff. 
Pronuclei,  15 

Radiation,  effect  on  spermatozoa, 

203 
Reciprocal  crosses,   190  ff.,   201 
Reighard,  J.  E.,  25,  141,  147 
Rejuvenation,  31,  34,  38 
Reversibility,  problem  of,  25,  128, 

161  ff. 
Richards,  A.,  27,  120,  121,  238 
Robertson,  T.  B.,  133,  232 
Roux,  W.,  64,  84,  85 
Ruckert,  J.,  79 

Schucking,  A.,  95,  134 
Schulze,  O.,  84 
Schwann,  Th.,  11 
Seeliger,  O.,  162 
Self-fertilization,  203  ff. 


Self-sterility,  26,  203  ff. 

Senescence,  t^t,,  34 

Serological  c<)m[)arisons,  26,  176, 
217.  219,  235,  236 

Sex  chromosomes,  68 

Shackell,  147,  148 

Shearer,  188,  193 

Smith,  J.  W.,  204 

Sobotta,  J.,  184 

Spallanzani,  Abbe,  6,  25 

Specificity,  problem  of,  26,  130, 
184  ff. 

Sperm:  aster,  60,  61,  73;  centro- 
some,  19,  49,  70,  71,  74;  nu- 
cleus, 16,  63,  71;  receptors,  27, 
236 

Spermatozoa:  activity,  92,  99, 
109,111,112;  agglutination,  of, 
27,  109,  112  ff.,  175,  220; 
aggregations,  94, .  102  ff.,  109, 
112;  behavior,  91  ff.,  96;  chemo- 
taxis,  102  ff.,  114;  discovery 
of,  4,  5;  duration  of  life,  loi, 
131,  132;  energy  production, 
92;  fertilizing  power  of,  131, 
132  ff.,  139;  fertilizing  sub- 
stance of,  132  ff.;  figures  of,  49; 
locomotion,  93;  mass  effect, 
134;  parts,  48;  path  of,  64,  65, 
84,  85;  penetration  of,  12,  16, 
22,  51,  53,  54,  64,  131,  267; 
reactions,  96  ff.;  thigmotaxis  of, 
98,  99,  107 

Stockard,  C.  R.,  160 

Stout,  205,  212,  214,  215 

Strongylocentrotus ,  1 1 1 ,  118,  1 24, 
148,  157,  164,  193 

Superposition  of  fertilization  and 
parthenogenesis,  163  ff. 


Teacher  (Bryce  and),  139 
Tennent,   D.    H.,    189,    iQO, 

^95 
Tissue  specificity,  1S4  ft. 

Toxopneusles^Gi,  14S 

Triepel,  A.,  139 

Van  Beneden  E.,  13,  15,  67 
Van  der  Stricht  O.,  76 
Vejdovsky,  F.,  56 
Vernon,  H.  M.,  189,  193 


193, 


278 


PROBLEMS  OF  FERTILIZATION 


Virchow,  R.,  17 
Viscosity  of  egg,  153 

Wagner,  12,  18,  19 
Waldeyer,  O.,  139 
Waldstein,  185 
Warburg,  0.,  145,  242 
Wastenys,  H.,  145 
Weismann,  A.,  38,  40 
Whitman,  C.  O.,  185 


Wierzejsky,  A.,  56 

Wilson,  E.  B.,  25,  49,  60,  61,  64, 

140,  158,  162,  163,  233,  244,  265 
Winkler,  H.,  133 
Woodruff,  L.  L.,  39,  238 
Woodward,  A.  E.,   27,   120,   121, 

231,  238,  239,  240 

Ziegler,  H.  E.,  158 
Zygote,  31 


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